Expression systems utilizing autolyzing fusion proteins and a novel reducing polypeptide

ABSTRACT

The present invention provides expression systems for exogenous polypeptides wherein the polypeptide is expressed as a fusion protein together with clover yellow virus Nuclear Inclusion a (NIa), the NIa component serving to autolyze the fusion protein after expression. This system can be used to express a novel polypeptide which we have designated KM31-7 protein and which is capable of reducing dichloroindophenol and reduced glutathione. This polypeptide is useful in the treatment of disorders caused by oxidative stress.

This is a division of application Ser. No. 09/167,151 filed Oct. 6, 1998(U.S. Pat. No. 6,307,038), which is a Divisional Application ofapplication Ser. No. 08/500,635 filed Jul. 11, 1995 (U.S. Pat. No.5,955,072).

FIELD OF THE INVENTION

The present invention relates to polypeptide expression systemsrequiring cleavage of a precursor product, and to proteases for use insuch systems. The present invention further relates to a novelpolypeptide capable of reducing dichloroindophenol and oxidizedglutathione, DNA encoding the novel polypeptide, vectors containing suchDNA, hosts transformed with such vectors, and pharmaceuticalcompositions containing the polypeptide. In addition, the presentinvention also relates to monoclonal antibodies against thispolypeptide, and a process for isolating and purifying the polypeptideusing such an antibody.

PRIOR ART

The Potyviruses are a group of viruses each of which have asingle-stranded, RNA genome of approximately 10,000 bases and whichinfects plants such as the family Solanaceae. The Potyvirus genome ischaracterized by possessing one extremely long open reading frame, orORF, [Dougherty, W. G. and Hiebert, E. (1980), Virology 101:466-474.;Allison, R. et al. (1986), Virology 154: 9-20]. In order to express theindividual proteins encoded within the ORF, the translated polyproteinis digested by two types of protease, both of which are also encodedwithin the ORF [Dougherty, W. G. and Carrington, J. C. (1988), Ann. Rev.Phytopath. 26: 23-143].

Tobacco etch virus (TEV) is a member of the Potyvirus family, and thisvirus produces nuclear inclusions which can be stained with trypan bluein the infected cell. The nuclear inclusions apparently consist of twokinds of protein, one of which has proven to be a viral protease, andwhich has been designated Nuclear Inclusion a, or NIa [J. Virol.,61:2540-2548 (1987)].

The Nuclear Inclusion a proteases of the Potyviruses recognize andcleave a peptide sequence which includes one of Gln-Gly, Gln-Ser andGln-Ala, and it is believed that this sequence is hexameric and occursat the C-terminal end of the relevant NIa within the polyprotein.Cleavage is between the two residues making up the dimers shown above.

The complete genomic sequences of TEV and tobacco vein mottling virus(TVMV), another member of the Potyvirus family, have been determined,and homology searching has allowed the location of the NIa's of theseviruses within their respective genomes [Virology, 154: 9-20 (1986);Nucleic Acid Res., 14:5417-5430 (1986)].

Clover Yellow Vein Virus, or CYVV, is also a Potyvirus. So far, only thegene occurring at the 3′ end of the CYVV genome, together with the coatprotein it encodes, has been sequenced [Uyeda, I. et al. (1991),Intervirol. 32: 234-245]. The structure of NIa region of the genome hasnot previously been elucidated, nor has the corresponding NIa beenisolated.

The production of exogenous proteins by expression systems can bestraightforward, using techniques well known in the art. However, thereare many polypeptides which cannot easily be expressed in an exogenoussystem. The problem may be that the polypeptide cannot be expressed inlarge amounts, and this cannot usually be corrected merely by placing aregulatory gene upstream. Alternatively, it may be thatpost-transcriptional events required to obtain the mature form do nottake place, or take place incorrectly.

For example, many eukaryotic polypeptides are initially translated withan N-terminal methionine which is subsequently deleted to obtain themature form. This processing cannot take place in prokaryotes, so thatalternative means of obtaining expression have had to be found. One suchtechnique involves fusing the desired exogenous protein withmaltose-binding protein or glutathione S-transferase, for example,purifying the expressed fusion protein and then cleaving with aprotease, such as Factor Xa, enterokinase, or thrombin. The maindrawback of this cumbersome method is that it requires two purifictionsteps, which results in a substantial loss of the end product.

U.S. Pat. No. 5,162,601 discloses the use of TEV protease in themanufacture of a polyprotein having linker sequences between each of theproteins it is desired to express, such as human tPA. However, thispatent only discloses the cloning of a multigene encoding thispolyprotein into a host. There is no disclosure of expression orpurification of the proteolytically cleaved end product.

Oxygen for metabolic energy is generally provided in the form ofoxidizing agents in the cellular environment. The activated form inwhich the oxygen is used is generally as a free radical, such assuperoxide (O₂ ⁻), peroxide (H₂O₂) or a hydroxy radical (OH⁻), all ofwhich are reduced to form water (H₂O) after use. Oxygen gas, itself, ishighly oxidizing, but the term “activated oxygen”, as used herein,relates to oxygen and oxygen-containing molecules which have greateroxidizing potential than atmospheric oxygen. The most potent form ofactivated oxygen is the free radical, which is a molecule or atom havingone or more unpaired electrons.

Free radicals are typically unstable and, if not properly controlled,can denature lipids, proteins and nucleic acids. Consequently, althoughactivated oxygen is essential to life, it is also a potential healthhazard, and must be very closely controlled. Even in vanishingly smallamounts, activated oxygen can cause disorders, due to high reactivity.As a result, living organisms are unable to survive unless they areequipped with a defence mechanism against activated oxygen.

In the cellular environment, the locations, amounts and times ofgeneration of activated oxygen must be carefully balanced against thecell's ability to neutralize the associated danger. This ability istypically provided by a defence mechanism that uses its own antioxidantsor antioxidation enzymes. In the context of the present invention, an“antioxidant” is the generic name for a naturally occurring substancewhich is able to prevent or inhibit the auto-oxidation of lipids, forexample. The term “antioxidation enzyme” is used generically for anenzyme which catalyzes a reaction which eliminates activated oxygen, theterm “antioxidative action” being construed accordingly.

Excessive amounts of activated oxygen are produced in a number ofabnormal circumstances, such as when a person is stressed, is takingdrugs, smokes, undergoes surgery, has an organ transplant or if hesuffers ischemia through a cerebral or myocardial infarction. Theselarge amounts are more than the control systems of the body caneliminate, so that the excess of activated oxygen can cause furtherdamage to the body, seriously impairing normal cells. The resulting,so-called oxidative stress is responsible for a great many diseaseconditions.

To take arteriosclerosis as an example, the occurrence of low specificgravity lipoproteins which have been oxidized by activated oxygen isconsidered to be one of the causes of the disease [Steinberg, D. (1983,)Arteriosclerosis 3, 283-301]. Oxidative stress is also considered to beintimately involved with cause and effect in the mechanisms associatedwith the occurrence, metabolic abnormalities and vascular complicationsof diabetes [Kondo, M. ed., “Approaches from Modern Medicine (4) FreeRadicals”, Medical View Pub., pp. 138-146].

Activated oxygen is also implicated in other pathological states andconditions, such as; ischemic disorders (reperfusion disorders, ischemicheart disease, cerebroischemia, ischemic enteritis and the like), edema,vascular hyperpermeability, inflammation, gastric mucosa disorders,acute pancreatitis, Crohn's disease, ulcerative colitis, liverdisorders, Paraquat's disease, pulmonary emphysema, chemocarcinogenesis,carcinogenic metastasis, adult respiratory distress syndrome,disseminated intravascular coagulation (DIC), cataracts, prematureretinopathy, auto-immune diseases, porphyremia, hemolytic diseases,Mediterranean anemia, Parkinson's disease, Alzheimer's disease,epilepsy, ultraviolet radiation disorders, radioactive disorders,frostbite and burns.

Several defence mechanisms exist both inside and outside cells for thesole purpose of eliminating activated oxygen generated physiologically.

Intracellularly, antioxidants and antioxidative enzymes, such as thosegiven below, are known to process and eliminate activated oxygen. Forexample, catalase is present in peroxisomes, and this enzyme reduces andremoves hydrogen peroxide. Glutathione peroxidase occurs in thecytoplasm and the mitochondria, and this enzyme reduces and detoxifieshydrogen peroxide and lipid peroxides in the presence of reducedglutathione. Transferrin, ferritin and lactoferrin, for example, inhibitthe generation of activated oxygen by stabilizing iron ions, whileceruloplasmin performs a similar function in connection with copperions. In addition, superoxide dismutase, which is present in thecytoplasm and mitochondria, catalyzes the reduction of superoxides toform hydrogen peroxide, the hydrogen peroxide then being eliminated bycatalase, for example. In addition, vitamins C and E, reducedglutathione and other low molecular weight compounds are also capable ofreducing and eliminating activated oxygen.

On the other hand, such agents as extracellular superoxide dismutase,extracellular glutathione peroxidase and reduced glutathione exist inthe extracellular environment, and these have similar modes of action totheir intracellular counterparts listed above. However, compared to theintracellular situation, there are fewer types of extracellularantioxidants and antioxidative enzymes, and there is only a small numberthat exhibit extracellular antioxidative action.

Reduced glutathione has an important function in maintaining the reducedstate both inside and outside cells, and it is represented by theformula below. Glutathione was first discovered in yeast byde-Rey-Pailhade in 1888, and it was later named glutathione followingits isolation as a compound by Hopkins in 1921.

Glutathione is composed of three amino acids:—glutamic acid, cysteineand glycine. The thiol groups of two glutathione molecules can beoxidized to form a disulfide bond in the presence of activated oxygen,thereby reducing the activated oxygen.

Glutathione is mainly produced in the liver, and circulates within thebody via the plasma. In the normal body, glutathione exists nearlyentirely in the reduced form. When levels of the oxidized form increase,then the reduced form is regenerated by the action of glutathionereductase in the presence of nicotinamide adenine dinucleotide phosphate(NADPH). Thus, reduced glutathione protects the cell membrane fromoxidative disorders and functions by the reducing activated oxygen andfree radicals. As a result of having this antioxidative property,reduced glutathione also protects against the effects of radioactivityand is useful as a therapeutic drug for cataracts. It has also recentlybeen reported that systemic levels of reduced glutathione are reduced inAIDS patients, which tends to indicate that the role of reducedglutathione in the body is extremely important. However, in abnormalconditions, the amount of activated oxygen can be so great thatvirtually all glutathione is in the oxidized state, so that activatedoxygen is not removed as fast as possible.

Human thioredoxin is a second example of a substance which exertsvarious physiological actions by means of its reducing activity, bothinside and outside cells. Human thioredoxin (also known as Adult T CellLeukemia Derived Factor, ADF) was cloned as a factor which was capableof inducing interleukin 2 receptors (IL-2R) in adult T cell leukemiacell lines. It is a thiol-dependent reductase with two cysteine residuesat its active site, and it is capable of reducing activated oxygen andfree radicals.

In addition to inducing IL-2 receptors, human thioredoxin also: promotescell growth in B cell strain 3B6 which is infected with Epstein-Barrvirus (EBV); protects against tumor necrosis factor (TNF) derived frommonocyte-origin cell line U937; and protects against vascularendothelial cell impairment by neutrophils. Further, in theintracellular environment, human thioredoxin acts on the transcriptionfactors NFkB, JUN and FOS through its reducing activity, thereby topromote DNA bonding activity and enabling it to function to increasetranscriptional activity. Human thioredoxin is currently being developedas a protective agent for radioactivity disorders, as well as for atherapeutic drug for reperfusion disorders, rheumatoid arthritis andinflammations, all of which disorders can be protected against by itsability to protect against cellular impairment via its reducingactivity.

As has been described above, it is physiologically extremely importantto maintain both the intracellular and extracellular environments in areduced state by elimination of activated oxygen and free radicals.There are believed to be many, as yet unknown, antioxidants andantioxidative enzymes, both inside and outside cells, that have a roleto play in removing activated oxygen and free radicals. Accordingly, itwould be extremely useful to find a reducing substance which was capableof regenerating, for example, reduced glutathione. Such a substancecould help in abnormal bodily conditions, such as those described above.

SUMMARY OF THE INVENTION

It is a first object of the present invention to provide a novelprotease, a nucleotide sequence encoding the protease, a vectorcontaining a DNA sequence encoding the protease, and a host cell whichhas been transformed with said vector.

It is a second object of the present invention to provide DNA encoding aprotein of interest and also encoding the novel protease upstream fromsaid protein, a sequence between the two said sequences further encodinga peptide cleavable by said protease, all of said sequences being in thesame open reading frame. It is also an object to provide a proteinencoded by said DNA, as well as a vector containing said DNA and anexpression system comprising said vector, said vector being able toreplicate autonomously in an appropriate host cell, such as bycomprising a nucleotide sequence required for autonomous replication.

In the alternative, it is a first object of the present invention toprovide a nucleotide sequence encoding a novel polypeptide havingreducing activity in vivo. It is also an object to provide such DNAwhich encodes a peptide which is capable of reducing dichloroindophenoland oxidized glutathione.

It is a further object of the present invention to provide a recombinantvector comprising the above-mentioned DNA, said vector being able toreplicate autonomously in an appropriate host cell, such as by having abase sequence enabling autonomous replication.

Moreover, it is a yet further object of the present invention to providea host cell microorganism which has been transformed with theabove-mentioned recombinant vector. It is also an object to provide theabove-mentioned peptide as an expression product from the transformedhost cell, and to provide a monoclonal antibody against the peptide.

We have now identified and cloned the novel CYVV protease (NIa) and havesurprisingly found that it is possible to use CYVV NIa as part of afusion protein which can be expressed in such hosts as E. coli and whichallows the production of large quantities of the fusion protein whichcan self-cleave to yield the desired exogenous protein. The CYVV NIagene can be stably maintained and expressed in Escherichia coli, and theexpressed NIa retains its activity as a specific protease, even when theprotein forms part of a fusion protein.

We have also discovered DNA that codes for a novel polypeptide which iscapable of reducing dichloroindophenol {also known asdichlorophenol-indophenol, 2,6-dichloroindophenol, or2,6-dichloro-4-[(4-hydroxyphenyl)imino]-2,5-cyclohexadien-1-one} andoxidized glutathione, said polypeptide being obtainable in large amountsby the use of appropriate genetic engineering techniques. Thispolypeptide is particularly useful in the therapy of conditions causedby, or related to, oxidative stress, or any disease caused by activatedoxygen, such as arteriosclerosis, diabetes and ischemic disorders(including reperfusion disorders, ischemic cardiac diseases,cerebroischemia and ischemic enteritis).

Thus, in a first aspect of the first embodiment of the presentinvention, there is provided a polynucleotide sequence wherein saidsequence comprises, in the 5′ to 3′ direction and in the same openreading frame:

a) a sequence encoding the clover yellow vein virus Nuclear Inclusion aprotein, or a mutant or variant thereof having similar proteolyticspecificity to that of clover yellow vein virus Nuclear Inclusion aprotein;

b) a sequence encoding a peptide recognizable by and cleavable by saidclover yellow vein virus Nuclear Inclusion a protein, or said mutant orvariant thereof; and

c) at least one sequence encoding a polypeptide.

The present invention also provides a sequence encoding the cloveryellow vein virus Nuclear Inclusion a protein, or a mutant or variantthereof having similar proteolytic specificity to that of clover yellowvein virus Nuclear Inclusion a protein.

The present invention further provides a vector, especially anexpression vector, containing a sequence as defined above.

The present invention still further provides a host transformed with avector as defined above, as well as an expression system comprising saidhost and said expression vector, and also a polypeptide produced by suchan expression system.

In the alternative embodiment of the invention, there is provided, in afirst aspect, a polynucleotide sequence encoding a polypeptide havingthe amino acid sequence of amino acid numbers 1 to 526 of sequence IDnumber 12, or which encodes a mutant or variant of said polypeptide,provided that the polypeptide encoded by the polynucleotide sequence iscapable of reducing dichloroindophenol and oxidized glutathione.

There is also provided a vector, especially an expression vector,containing a sequence as defined above.

The present invention still further provides a host transformed with avector as defined above, as well as an expression system comprising saidhost and said expression vector, and also a polypeptide produced by suchan expression system.

The invention also provides the above polypeptide for use in therapy, aswell as the use of such a polypeptide in the treatment and prophylaxisof conditions caused by, or related to, oxidative stress, or any diseasecaused by activated oxygen, and pharmaceutical compositions comprisingthe polypeptide.

There is yet further provided an antibody, especially a monoclonalantibody, and equivalents thereof, against the polypeptide, and theinvention additionally provides a method of producing such an antibodyand a method of purification of the polypeptide using the antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention will be illustrated with respect to theaccompanying drawings, in which:

FIG. 1 is a restriction enzyme map of cDNA of the NIa region isolatedfrom CYVV-cDNA;

FIG. 2 is a schematic diagram which shows the construction of plasmidpKNI5′ containing a 5′ region of NIa;

FIG. 3 is a schematic diagram which shows the construction of plasmidpKNI5IL containing a part of the IL-11 gene and a 5′-region of NIa;

FIG. 4 is a schematic diagram which shows primers which were used toprepare the 5′IL DNA fragment, the CIN3 DNA fragment and in which the3′-terminus of the NIa gene and the 5′-terminus of the IL-11 gene arefused;

FIG. 5 is a schematic diagram which shows the fusion of the CIN3 DNAfragment and the 5′IL DNA fragment by PCR;

FIG. 6 is a schematic diagram which shows the construction of plasmidpKSUN9;

FIG. 7 is a construction enzyme map of pUCKM31-7;

FIG. 8 is a comparative diagram of the nucleotide sequences of the 3′terminals in pUCKM31-7 and pcD-31;

FIG. 9 is a construction diagram of pSRα31-7;

FIG. 10 is a schematic diagram showing the introduction of a histidinehexamer encoding sequence into pUCKM31-7;

FIG. 11 is a construction diagram for pMAL31-7;

FIGS. 12A and 12B are graphs showing the results of the assay ofdichlorophenol-indophenol reducing activity; and

FIG. 13 is a graph showing the determination of oxidized glutathionereducing activity.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be illustrated firstly by reference to thefirst embodiment of the present invention, but the following discussionis also appropriate to the second embodiment of the invention, unless itis clear that the discussion is not applicable to the second embodiment.

It will be appreciated that it is preferred for the polynucleotidesequence of the invention generally to be in the form of DNA, andreferences hereon in will generally be to DNA, for this reason. However,such references also include RNA, where appropriate. RNA is not sopreferred, as uses therefor are limited by practicality. For example,mRNA can be expressed in xenopus oocytes or a wheatgerm lysate system,but neither of these is practical for producing large amounts of thefusion protein on an economic basis.

As used herein, the term “peptide” means any molecule comprising 2 ormore amino acids linked via a peptide bond. As such, the term includesoligopeptides, polypeptides and proteins. The term “fusion protein”relates to any single polypeptide obtained by combining two or moreother peptide sequences.

It will also be appreciated that the sequence of the invention ispreferred to be in the form of a double strand, and that the presentinvention also envisages the antisense sequence corresponding to thesequence of the invention. The double strand (or ds) sequence of theinvention may have one or two “sticky” ends, in which case the antisenseand sense strands will not necessarily correspond exactly.

The protein encoded by the sequence identified in a) above is intendedto cleave the peptide encoded by the sequence of b) above in order torelease the polypeptide(s) encoded by the sequence of c) above when thesequence of the invention is expressed in a suitable expression system.

Cleavage of the fusion protein may take place at any time after thesequences of a) and b) above have been translated. As such, it will beappreciated that the polypeptide encoded by c) above need not have beenfully translated before cleavage. However, in practice, we have foundthat at least some of the fusion protein, if not the majority, is fullytranslated prior to cleavage.

Where the sequence of c) above encodes more that one polypeptide, thenit is necessary to encode further cleavage sequences between eachencoded polypeptide, unless it is desired, for example, to obtain anuncleaved, fused plurality of polypeptides.

If the sequence of c) above encodes more than one polypeptide, then thesequence may be susceptible to attenuation in a transcriptionenvironment. In the process of attenuation, transcription of the mRNAsequence of the invention stops before the whole of the mRNA has beenread, so that polypeptides encoded nearer the 3′ end of the sequencewill be produced in smaller amounts than those encoded nearer to the 5′end. Attenuation is not generally a problem, except where severalpolypeptides are encoded and/or the polypeptides are very long.

It is also generally preferred for the sequence of c) above to encodeonly one polypeptide, unless it is desired to prepare a plurality ofpeptides for use together, or where such a plurality is fused.Otherwise, it is necessary to isolate the individual polypeptides aftercleavage, and this can be cumbersome and waste the end product throughpurification procedures.

However, where the sequence in c) above encodes more than onepolypeptide, and there is a cleavage sequence between each encodedpolypeptide, then the cleavage sequence does not necessarily have torecognized by CYVV NIa. All that is necessary is for the cleavagesequence between the sequence of a) and the 5′ end of the sequence of b)to be recognized by the protease encoded by the sequence of a). Where itis desired or acceptable to allow the fusion protein to self-digest toprepare a plurality of polypeptides, then any further cleavage sequencesmay be recognizable by the protease encoded by the sequence of a).However, such further sequences may be selected so that they arecleavable by other means, so that the fusion protein is cleaved by itsNIa portion after transcription to yield NIa and a polyprotein. The NIacan then be removed and the polyprotein cleaved by, for example, FactorXa or trypsin.

The cleavage peptide encoded by the sequence of b) above (also referredto herein as “cleavage sequence” and “cleavage peptide”) may be asequence which is wholly or partly comprised in either of the sequencesencoded for by the sequences of a) and c) (“NIa” or “protease”, and“polypeptide”, respectively). Thus, the N-terminal end of the cleavagepeptide may also be included in the C-terminal sequence of the protease,while the the C-terminal portion of the cleavage peptide may be includedin the N-terminal portion of the polypeptide. In such an instance, thecleavage peptide has no independent existence, and the recognition sitefor the protease is made up from the C-terminal of the protease and theN-terminal of the polypeptide.

The cleavage peptide may also be included in only part of either theprotease or the polypeptide. In such an instance, the N-terminal of thecleavage peptide will normally be included in the C-terminal portion ofthe protease, and the N-terminal portion of the polypeptide will eitherbe linked directly to the cleavage sequence or there may be one or moreamino acid residues between the polypeptide and the linker.

Where there are one or more amino acid residues between the cleavagesequence and the protease and/or the cleavage sequence and thepolypeptide, then the number and nature of such residues should be suchas not to prevent the action of the protease. Excess amino acid residueson the N-terminal of the polypeptide will not generally be advantageouswhere such residues have to be removed in order to obtain the matureform of the polypeptide. It is generally preferred, where possible, andin the absence of contraindications, to engineer the cleavage peptide sothat the mature form of the desired protein is obtained on cleavage ofthe fusion protein. However, it is entirely possible to obtain a proteinhaving Gly, Ser or Ala attached to the N-terminal for example, and thesecan be removed by the action of a suitable aminopeptidase, if desired.

In some instances, it may be desirable to encode a proline between theN-terminal of the polypeptide and the cleavage sequence. This allowscleavage of residues up to the proline residue by the action ofaminopeptidase P (3.4.11.9), for example, but not beyond. Instead, theproline residue can then be removed by the action of prolineiminopeptidase to yield the mature protein. This forms a preferredembodiment of the invention.

The sequence of a) encodes a protease that is capable of cleaving thefusion protein encoded by the sequence of the invention. In the presentinvention, this protease is clover yellow vein virus Nuclear Inclusion aprotease, or a mutant or variant thereof having similar proteolyticspecificity to that of clover yellow vein virus Nuclear Inclusion aprotease.

The clover yellow vein virus NIa protease is encoded by nucleotidenumbers 10 to 1311 in sequence ID number 1 in the sequence listing,while the primary sequence of NIa is given by amino acid numbers 4 to437 in sequence ID number 2 in the sequence listing. These sequences arenovel, and form a part of the present invention, as do mutants andvariants thereof.

Nuclear Inclusion a has proteolytic activity which specificallyhydrolyzes the peptide bond between Gln-Ala, Gln-Gly or Gln-Ser in asubstrate peptide. In addition, we have also found that NIa can cleaveGln-Val. Thus, in contrast to other proteases of the Potyvirus family,we have found that CYVV NIa (references to NIa herein should be taken asmeaning CYVV NIa, unless otherwise specified) can cleave the sequenceAsnCysSerPheGlnX, wherein X is any amino acid residue, but especiallyGly, Ala, Val or Ser, and particularly Gly, Ala or Ser.

It will also be appreciated that peptides comprising the sequenceAsnCysSerPheGlnX can be cleaved by NIa, especially where such sequenceis sterically exposed to the action of NIa. Thus, the present inventionalso provides a system for the preparation of a polypeptide, wherein aprecursor form of the polypeptide containing the sequenceAsnCysSerPheGlnX is cleaved by NIa. This system may also comprise otherprocessing steps, as appropriate, either before, after orcontemporaneously with the cleavage by NIa.

Although we generally prefer to use a sequence encoding naturallyoccurring NIa (in accordance with Sequence ID 2), the present inventionequally contemplates the use of mutants or variants of NIa.

As stated, the protease should have the same or similar specificity asthat of naturally occurring NIa. In this respect, the protease shouldgenerally share substantial sequence homology with amino acid residues 4to 437 in sequence ID number 2 except where it is apparent to oneskilled in the art that substantial variation is possible withoutchanging the recognition sequence or reducing activity below usefullevels.

In general, it will be appreciated that the activity of any givenprotein is dependent upon certain conserved regions of the molecule,while other regions have little importance associated with theirparticular sequence, and which may be virtually or completely redundant.Accordingly, as stated above, the present invention includes anyvariants and mutants on the sequence which still show a specificitysimilar to that of naturally occurring NIa. Such variants and mutantsinclude, for example, deletions, additions, insertions, inversions,repeats and type-substitutions (for example, substituting onehydrophilic residue for another, but not strongly hydrophilic forstrongly hydrophobic as a rule). Small changes will generally havelittle effect on activity, unless they are in an essential part of themolecule, and such changes may be as a side-product of geneticmanipulation, for example, when generating extra restriction sites, ifsuch is desired.

In general, there will not usually be any particular reason to want tochange the structure of NIa, except in circumstances apparent to thoseskilled in the art. Indeed, most mutations and variations to either theamino acid or coding sequence will be as a result of the isolation ofnovel variations on the original wild-type virus. Neverthelessdeliberate, and even accidental, modifications are not excluded from thepresent invention, provided that the protease has the necessaryspecificity and sufficient proteolytic activity.

As used herein, the term “adverse effect” means any effect onspecificity or activity which renders the protease significantly lesseffective than the naturally occurring NIa identified above, to theextent that activity is reduced below a useful level.

Many substitutions, additions, and the like may be effected, and theonly limitation is that activity not be adversely affected. In general,an adverse effect on activity is only likely if the 3-D (tertiary)structure of the NIa is seriously affected.

The term “mutants” is used herein with reference to deletions,additions, insertions, inversions and replacement of amino acid residuesin the sequence which do not adversely affect activity. The presentinvention further includes “variants”, this term being used in relationto naturally occurring CYVV NIa which corresponds closely to sequence ID2, but which varies therefrom in a manner to be expected within a largepopulation. Within this definition lie allelic variation and thosepeptides from other cultivars showing a similar type of activity andhaving a related sequence.

It will be appreciated that neither the NIa nor the protein of thesecond embodiment of the present invention needs to correspondcompletely to the sequences depicted in Sequence ID's 2 and 12respectively. The only requirement is that each has the desiredactivity, even if the polypeptide is only a fraction of the whole of thenatural sequence, or even a mutant or variant of such a fraction.

The genes of eukaryotes, such as the interferon gene, are generallyconsidered to demonstrate polymorphism [c.f., Nishi, T. et al. (1985),J. Biochem. 97, 153-159]. This polymorphism results in some cases whereone or more amino acids are substituted in a polypeptide, as well asother cases where there are no changes in the amino acid sequence,despite substitution of the nucleotide sequence.

Thus, it will be appreciated that the polynucleotide coding sequence mayalso be modified in any manner desired, provided that there is noadverse effect on protease activity. Spot mutations and other changesmay be effected to add or delete restriction sites, for example, tootherwise assist in genetic manipulation/expression, or to enhance orotherwise conveniently to modify the NIa molecule.

The terms “mutant” and “variant” are also used herein with reference tothe polynucleotide sequence, and such references should be construed inan appropriate manner, mutatis mutandis. It will be appreciated that,while a mutant or variant of a peptide sequence will always be reflectedin the coding nucleotide sequence, the reverse is not necessarily true.Accordingly, it may be possible for the nucleotide sequence to besubstantially changed (see discussion of degeneracy of the genetic codebelow), without affecting the peptide sequence in any way. Such mutantsand variants of the nucleotide sequence are within the scope of theinvention.

For example, it has been established that the protein obtained byreplacing a cysteine codon in the interleukin 2 (IL-2) gene with aserine codon is still capable of expressing IL-2 activity [Wang, A. etal. (1984), Science 224: 1431-1433]. Therefore, as long as a sequenceencodes a naturally occurring or a synthetic protein having theappropriate NIa activity, then it is included within the presentinvention.

A gene encoding the NIa of the invention may easily bereverse-engineered by one skilled in the art from sequence ID 2.

It will be appreciated that any one given reverse-engineered sequencewill not necessarily hybridize well, or at all, with any givencomplementary sequence reverse-engineered from the same peptide, owingto the degeneracy of the genetic code. This is a factor common in thecalculations of those skilled in the art, and the degeneracy of anygiven sequence is frequently so broad as to make it extremely difficultto synthesise even a short complementary oligonucleotide sequence toserve as a probe for the naturally occurring oligonucleotide sequence.

The degeneracy of the code is such that, for example, there may be 4, ormore, possible codons for the most frequently occurring amino acids.Accordingly, therefore, it can be seen that the number of possiblecoding sequences for any given peptide can increase exponentially withthe number of residues. As such, it will be appreciated that the numberof possible coding sequences for the NIa of the invention may have sixor more figures. However, it may be desirable to balance the G+C contentaccording to the expression system concerned, and other factors such ascodon frequency for the relevant expression system should generally betaken into account.

As stated above, hybridization can be an unreliable indication ofsequence homology but, nevertheless, those sequences showing in excessof 50%, preferably 70% and more preferably 80% homology with sequence ID1 are generally preferred. In each case, it will be appreciated that aprotease, as defined, should be encoded.

The present invention also envisages the possible use of a leadersequence encoded upstream of the protease. This would allow the fusionprotein to be externalized from the host, and collected in the culturesupernatant, for example. The resulting externalized fusion proteinwould then be allowed to self-digest, and the polypeptide collected. Forsuch signal sequences, any suitable sequence may be used, especiallywhere such has been specifically developed for a given expressionsystem.

The present invention also envisages vectors containing the sequence ofthe present invention. The general nature of vectors for use inaccordance with the present invention is not crucial to the invention.In general, suitable vectors and expression vectors and constructionstherefor will be apparent to those skilled in the art, and will bechosen according to precisely what the practitioner wishes to achievewith the sequence, such as cloning or expression.

Suitable expression vectors may be based on ‘phages or plasmids, both ofwhich are generally host-specific, although these can often beengineered for other hosts. Other suitable vectors include cosmids andretroviruses, and any other vehicles, which may or may not be specificfor a given system. Suitable control sequences, such as recognition,promoter, operator, inducer, terminator and other sequences essential toand/or useful in the regulation of expression, will be readily apparentto those skilled in the art, and may be those associated with CYVV orwith the vector used, or may be derived from any other source assuitable. The vectors may be modified or engineered in any suitablemanner.

It will be appreciated that sequence ID 2 represents sufficient sequenceto encode entire NIa. Terminators, promoters and other such controlsequences as desired may be added so as to, for example, facilitateligation into a suitable vector, or expression, or both.

It will be appreciated that a DNA fragment encoding the NIa of theinvention, together with any fragment encoding the cleavage sequence andthat encoding the polypeptide(s) may easily be inserted into anysuitable vector. Ideally, the receiving vector has suitable restrictionsites for ease of insertion, but blunt-end ligation, for example, mayalso be used, although this may lead to uncertainty over the openreading frame and direction of insertion. In such an instance, it is amatter of course to test hosts transformed with the transfected vectorto select vectors having the necessary fragments inserted in the correctdirection and in the correct ORF. In order to ensure that the fragmentsare in the correct ORF, it may be desirable to create a construct of thesequences a), b) and c) which can then be inserted directly into thevector, thereby reducing the uncertainty of obtaining the desiredexpression vector. Suitable vectors may be selected as a matter ofcourse by those skilled in the art according to the expression systemdesired.

By transforming E. coli, for example, with the plasmid obtained,selecting the transformant with ampicillin or by other suitable means,and adding tryptophan or other suitable promoter inducer (such asindoleacrylic acid), the desired fusion protein may be expressed. Theextent of expression may be analyzed by SDS polyacrylamide gelelectrophoresis—SDS-PAGE [Laemmli et al., Nature, (1970), 227,pp.680-685].

It will also be appreciated that, where another vector is used, forexample, it will be equally acceptable to employ any suitable selectionmarker or markers, or an alternative method of selection, and/or to useany suitable promoter as required or convenient.

After cultivation, if the fusion protein is to be collected from thehost cells, then the transformant cells are suitably collected,disrupted, for example sonicated, and spun-down. Disruption may also beby such techniques as enzymic digestion, using for example cellulase, orby shaking with an agent such as glass beads, but methods such assonication are generally preferred, as no extra ingredients arenecessary. The resulting supernatant may be assayed for polypeptideactivity and the cleavage products can be determined by SDS-PAGE, forexample.

Conventional protein purification is suitable to obtain the expressionproduct.

The DNA of the present invention may be prepared by isolating the RNAgenome from clover yellow vein virus [Uyeda, I. et al. (1975) Ann.Phytopath. Soc. Japan 41: 92-203]. A suitable source of CYVV is AmericanType Culture Collection No. PV 123. In any event, clover yellow veinvirus may be defined as a virus which causes necrosis in Vicia faba.

Genomic RNA may be obtained from CYVV particles which have been purifiedfrom an infected plant and then reverse transcribed and double-strandedcDNA prepared by known methods.

For the purposes of determining whether a virus is a mutant or variantof the same strain of CYVV, sequence homology is a good indicator. Manytypes of Potyvirus are known, and they all vary in pathogenicity.Whether a given virus forms a separate member of the family is based onthe serological relation of the viral coat proteins and on the homologyof the amino acid sequences. Accordingly, viruses which have primaryamino acid sequences which share 90% homology are considered to be thesame strain, while viruses which have primary amino acid sequences whichshare less than 70% homology are considered to distinct family members[Shukla, D. D. and Ward, C. W. (1989), Arch. Virol. 106: 171-200]. Basedon the various properties of the coat protein of CYVV used in thepresent invention [Uyeda, I. et al. (1991), Intervirol. 32: 234-245],CYVV is recognized as an independent member of the Potyvirus family.Therefore, any virus having a coat protein primary amino acid sequencehomology of 90% or more, or any virus detecting as positive by ELISAusing an anti-CYVV anti-serum (for example, American Type CultureCollection No. PVAS-123: clover yellow vein virus antiserum), is definedas being a strain of clover yellow vein virus.

The various plant species upon which CYVV can be grown include;Phaseolus vulgaris, Vicia faba and Pisum sativtum, but Vicia faba,especially Vicia faba cultivar Wase-soramame, is preferred.

The preferred method for purifying the virus particle involveshomogenizing and squeezing a leaf or leaves of an infected plant in asuitable buffer, followed by extraction with an organic solvent, such aschloroform, and repeating differential centrifugation, followed bysucrose density gradient centrifugation.

One way to confirm that the virus particle thus obtained is CYVV can beperformed by examining the virus particle under an electron microscope.Another is by inoculating Vicia faba with the virus particle in order toobserve whether any symptoms occur.

Suitable methods for extracting the genomic RNA from virus particlesinclude the guanidinium thiocyanate/phenol method, the guanidiniumthiocyanate/trifluoro-cesium method and the phenol/SDS method. However,we prefer to use the alkaline sucrose density gradient centrifugationmethod [Dougherty, W. G. and Hiebert, E. (1980) Virology 101: 466-474].

The RNA obtained as described above can then be tested to confirm thatit indeed encodes a protease by translation in a cell-free translationsystem. Autolysis (self-digestion) can then be detected where the RNAcodes for more than just the protease in the total translation product,by monitoring any change in the molecular weights of harvested productsfrom the lysates. Such monitoring can be performed by means of ananti-coat protein antibody, for example.

Should it be required, then the production of translation product can bemonitored with passage of time using an anti-coat protein antibody, forexample, with the genomic RNA being translated by injection into Xenopuslaevis oocytes [Gurdon, J. B. (1972), Nature 233: 177-182], or by usinga rabbit reticulocyte or a wheat germ lysate system [Schleif, R. F. andWensink, P. C. (1981), in “Practical Methods in Molecular Biology”Springer-Verlag, N.Y.].

Single stranded DNA can be synthesized by using the thus obtainedgenomic RNA as a template using a reverse transcriptase, and ds-cDNA canbe synthesized from the single-stranded (ss) cDNA by following standardprocedures. Suitable methods include the S1 nuclease method[Efstratidiatis, A. et al. (1976), Cell 7: 279-288: Okayama, H. andBerg, P. (1982), Mol. Cell. Biol. 2: 161-170 and others], the Landmethod [Land, H. et al. (1981) Nucleic Acids Res. 9, 2251-2266] and theO. Joon Yoo method [Yoo, O. J. et al. (1983) Proc. Natl. Acad. Sci. USA79, 1049-1053]. Of the various options, we prefer to use theGubler-Hoffman method [Gubler, U. and Hoffman, B. J. (1983), Gene 25:263-269].

The thus obtained ds-cDNA can then be incorporated into a cloningvector, such as a plasmid or lamdha phage, and the resulting recombinantvector then transformed into a microorganism, such as Escherichia coli.E. coli strain DH5α is particularly preferred. Transformants can then beselected by their resistance to bactericidal agents, such astetracycline or ampicillin by techniques well known in the art.

Transformation can, for example, be carried out by the Hanahan method[Hanahan, D. (1983), J. Mol. Biol. 166: 557-580], wherein therecombinant DNA vector is introduced into a cell which has been madecompetent by treatment with calcium chloride, magnesium chloride orrubidium chloride.

Suitable methods for selecting transformants having NIa DNA are as shownbelow.

(1) Screening with a Probe

If it is desired to start from wild-type CYVV, then one way to isolatethe appropriate RNA is to use a cDNA probe, given that the amino acidsequence of NIa has been elucidated (the portion of the sequence usedmay be from any region of NIa). Thus, an oligonucleotide correspondingto the relevant amino acid sequence is synthesized. In general, theamino acid sequence chosen will involve the least amount of degeneracypossible, otherwise it will be necessary to produce several probes usingthe various codons possible. In such an instance, it is likely to be ofassistance to take codon usage frequency into consideration.Alternatively, plural nucleotide sequences can be considered, andinosine can be used to replace nucleotides which vary. The probe canthen be labeled with a radioisotope, such as ³²P, ³⁵S or biotin.Transformant strains can then be detected by fixing denatured plasmidDNA on nitrocellulose filters using the radiolabelled probes, positiveclones being detectable by autoradiography.

(2) Using a PCR Probe

In this technique, oligonucleotides both from the sense strand and fromthe anti-sense strand corresponding to a portion of the known amino acidsequence can be synthesized and the polymerase chain reaction [Saiki, R.K. et al. (1988), Science 239, 487-491] carried out. These can then beused in combination to amplify a DNA fragment encoding NIa. Suitabletemplate DNA is cDNA obtained through reverse transcription of viralgenomic RNA known to encode NIa. The thus prepared DNA fragments arelabeled, such as with ³²P, ³⁵S or biotin, and colony hybridization orplaque hybridization is carried out with this probe to select the cloneof interest.

(3) Screening by Exogenous Production in an Animal Cell

This method involves culturing a transformant strain to amplify a gene,followed by transfecting the gene into an animal cell (generally usingeither a plasmid which is replication competent and containing atranscription promoter region, or using a plasmid which can beintegrated into an appropriate chromosome) to express a protein encodedby the gene. By measuring the relevant activity, a strain can beselected.

(4) Selection Using an Antibody Against NIa

Antibody is produced against the nuclear inclusions (NIa and NIb) from aplant infected with CYVV, or is produced against a protein produced byan expression vector in an appropriate system. The antibody, or itsanti-antibody, can then detect the desired NIa or strain of interest.

(5) Selective Hybridization Translation System

Transformant cDNA is hybridized with mRNA from cells which express NIa,as described above, and the mRNA bound to the cDNA is dissociated andrecovered. The recovered mRNA is translated into protein in atranslation system, for example, by injection into Xenoous laevisoocytes, or into such cell-free systems as rabbit reticulocyte lysate orwheat germ lysate. The strain of interest is selected by examining theactivity of NIa or by detecting NIa by means of an antibody to NIa.

The DNA encoding CYVV NIa can be obtained from the transformant strainsof interest by known methods [c.f. Maniatis, T. et al. (1982), in“Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory,N.Y.]. For example, a fraction which corresponds to a vector DNA isseparated from the cells, and the DNA region which codes for saidprotein is excised from said plasmid DNA.

Determination of the thus obtained DNA sequence can be carried out byusing, for example, the Maxam-Gilbert chemical modification method[Maxam, A. M. and Gilbert, W. (1980), in “Methods in Enzymology” 65:499-2761, or by using, for example, a dideoxynucleotide chaintermination method using the M13 phage [Messing, J. and Vieira, J.(1982), Gene 19: 269-276].

In recent years, fluorochromes have tended to replace the use of themore dangerous radioisotope for the determination of DNA sequences. Inaddition, dideoxynucleotide chain termination is now generally performedby a robot under computer control. Systems which read base sequencesafter electrophoresis are also proliferating, and examples include the“CATALYST 800” sequencing robot and the 373A DNA sequencer (Perkin-ElmerJapan Applied Biosystems). These systems enable DNA base sequencedetermination procedures to be performed both efficiently and safely.

The vectors of the present invention can generally be so organized thatthey can be expressed in “standard cells”, either prokaryotic oreukaryotic. In addition, by introducing an appropriate promoter and asequence for phenotypic expression into the vector, the gene can beexpressed in assorted host cells.

Suitable prokaryotic hosts include Escherichia coli and Bacillussubtilis. For phenotypic expression of the relevant gene, the vector cansuitably contain a replicon originating from a species which iscompatible with the host. In E. coli, a plasmid might contain areplication origin and a promoter sequence such as lac or UV5. Vectorswhich can confer selectivity based on phenotypic character (phenotype)to a transformed cell are preferred.

Escherichia coli is often used as the host, and strain JM 109 derivedfrom E. coli strain K12 is a preferred host. Vectors for E. coli are,presently, generally selected from pBR322 or the pUC series of plasmids,but other strains and vectors can be used, as desired. Suitablepromoters for Escherichia coli include the lactose promoter (lac), thetryptophan promoter (trp), the tryptophan-lactose (tac) promoter, thelipoprotein (lpp) promoter, the lambda (λ) PL promoter (from λ phage)and the polypeptide chain elongation factor Tu (tufB) promoter, but thepresent invention is not limited to these promoters.

Suitable strains of Bacillus subtilis include strain 207-25. Suitablevectors include pTUB228 (Ohmura, K. et al. (1984), J. Biochem. 95:87-93] and others. A suitable promoter is the regulatory sequence of theBacillus subtilis α-amylase gene. This is an appropriate example whereina signal peptide sequence (from α-amylase), can be used forextracellular secretion.

If it is desired to express the fusion protein in eukaryotic cells, thencells from vertebrates, insects, yeasts, plants, etc. may be used. Thepreferred vertebrate cells are COS cells, especially COS-1 cells [e.g.Gluzman, Y. (1981), Cell 23: 175-182], or a Chinese hamster ovary cellline (CHO) deficient in dihydrofolate reductase [Urlaub, G. and Chasin,L. A. (1980), Proc. Natl. Acad. Sci. USA 77, 4216-4220], although thepresent invention is not limited to these.

As stated above, COS cells are suitably employed as vertebrate hostcells, and these may be used as an example. Expression vectorscontaining an SV40 replicon are able to replicate autonomously in COScells, and these are provided with a transcription promoter, atranscription termination sequence and one or more splicing sites.Expression vectors containing the desired DNA sequence can be used totransform COS cells by various methods, such as, for example, theDEAE-dextran method [Luthman, H. and Magnusson, G. (1983), Nucleic AcidsRes. 11, 1295-1308], the calcium phosphate-DNA coprecipitation method[Graham, F. L. and van der Eb, A. J. (1973), Virology 52, 456-457] andthe electroporation method [Neumann, E. et al. (1982), EMBO J. 1,841-8451.

If CHO cells are used as the host cells, then it is appropriate to use avector capable of expressing the neo gene which serves to provide G418resistance. Suitable vectors carrying this marker include pRSVneo[Sambrook, J. et al. (1989): “Molecular Cloning—A Laboratory Manual”,Cold Spring Harbor Laboratory, NY] and pSV2-neo [Southern, P. J. andBerg, P. (1982), J. Mol. Appl. Genet. 1, 327-341]. Transformants canthen be selected by their resistance to G418.

The selected transformant can be cultured by conventional methods and,in the case of the second embodiment of the invention, polypeptide isproduced both intracellularly and extracellularly. Suitable culturemedia can be selected from those commonly used, in accordance with thehost cells employed. For example, for COS cells, a medium containingblood components, such as fetal bovine serum, can be added as necessaryto media such as RPMI-1640 medium or Dulbecco's modified Eagle's medium(DMEM).

Should the practitioner prefer to use insect cells, then cells derivedfrom Spodoptera frugiverda [Smith, G. E. et al. (1983), Mol. Cell. Biol.3: 2156-2165] may be used.

Suitable yeasts include baker's yeast (Saccharomyces cerevisiae) andfission yeast (Schizosaccharomyces pombe).

Suitable plant cells include those from Nicotiana tabacum and Oryzasativa.

It will be appreciated that the hosts enumerated above are standardhosts in the art, and that the skilled person in the art will be able tochoose amongst these and other hosts as appropriate.

Suitable expression vectors for vertebrate cells include those whichhave a promotor located upstream from the gene to be expressed, togetherwith such sites as an RNA splicing site, a polyadenylation site, and atranscription-termination sequence, and further having a replicationorigin, if required. A suitable example of such an expression vector ispSV2dhfr, which has the SV40 early promotor [Subramani, S. et al.(1981), Mol. Cell. Bio. 1: 854-864].

A suitable expression system for insect cells includes cultured cells ofSpodoptera frugiperda. Suitable expression vectors have, for example,the Baculovirus Polyhedrin promoter located upstream from the gene to beexpressed, together with a polyadenylation site and a portion of theAcMNPV (Acutogranha californica nuclear polyhedrosis virus) genomerequired for homologous recombination. One example is pBacPAK8 [Matuura,Y. et al. (1987), J. Gen. Virol. 68: 1233-1250].

For eukaryotic expression, yeast is commonly used, such as baker's yeast(S. cerevisiae). Suitable expression vectors for yeast may include thealcohol dehydrogenase promoter [Bennetzen, J. L. and Hall, B. D. (1982),J. Biol. Chem. 257: 3018-3025], the acidic phosphatase promoter[Miyahara, A. et al. (1983), Proc. Natl. Acad. Sci. USA 80, 1-5], or thecarboxypeptidase Y promoter [Ichikawa, K. et al. (1993), Biosci.Biotech. Biochem. 57: 1686-1690], for example. In such an instance, thesignal peptide sequence from carboxypeptidase Y may also be used, inorder to effect secretion to the extracellular space.

Suitable expression vectors for plants include, for example, pBI121which has a ³⁵S promoter (derived from the early promoter of cauliflowermosaic virus), the polyadenylation sequence of the nopaline synthesisgene from Agrobacterium tumefaciens, and the Agrobacterium tumefaciensgene transfer sequence [Jefferson, R. A. et al. (1987), EMBO J. 6:3901-39071. Such vectors can be introduced into plant cells by suchmethods as infection with Agrobacterium tumefaciens and electroporation.

Plasmid pKK388-1 (manufactured by Clonetech Co.) has a trc promoter andis suitable for use in Escherichia coli. This expression vector is ableto autonomously replicate in a strain derived from Escherichia colistrain K12, such as strain JM109. This vector can easily be introducedinto Escherichia coli by such well known methods as are mentioned above.The thus obtained strain can be inoculated into a medium, such as thewell known LB medium, and cultured for a while.

The trc promoter can then be activated by adding, for example,isopropyl-β-galactopyranoside (IPTG), to induce the promoter. Afterfurther culturing, the expressed protein then can be extracted from thecells by disrupting the cells, such as with a sonicator. If it isdesired to produce a protein having Pro at the N-terminus, then it willgenerally be appropriate to use Escherichia coli as the host.

By applying the above description and, if necessary, taking into accountthe accompanying Examples, a fraction from the culture of suitablytransformed cells containing the desired polypeptide can be isolated andpurified by known methods, depending on the physical and chemicalproperties of the polypeptide. Suitable such methods include treatmentwith a protein precipitant, ultrafiltration, various chromatographies(such as molecular sieve chromatography (gel filtration), adsorptionchromatography, ion exchange chromatography, affinity chromatography andhigh performance liquid chromatography (HPLC)], dialysis andcombinations of the above methods.

In order to determine whether the expressed NIa protein has thenecessary protease activity, the purified NIa protein may be reactedwith a substrate protein containing a cleavage sequence for theprotease, such as the expression product of the gene encoding the fusionprotein which includes the viral coat protein (the natural substrate ofNIa) together with nuclear inclusion b (NIb) (Dougherty, W. G. et al.(1988), EMBO J. 7: 1281-1287]. Such a fusion protein may be isolatedfrom the viral genomic cDNA and inserted into an expression vector. Theresulting expression vector is then introduced into a suitable host cellto produce the fusion protein. By treating the resultant fusion proteinin known manner, such as with a protein precipitant or bychromatography, the fusion protein can be separated and purified.

The thus separated and purified substrate protein can then be reactedwith said protease in a suitable buffer solution at a suitabletemperature to recover a reaction product. The recovered reactionproduct can then be subjected to electrophoresis and to Western blottingusing an anti-coat protein antibody to allow the protease activity to bedetected as a difference in the mobility of a band. In this method, itis also possible for a a synthetic oligopeptide containing anappropriate cleavage sequence to be used.

The proteolytic activity of the protease of the present invention can bedetected without purification. Thus, activity can be measured byconnecting the protease gene in sequence and in the same open readingframe, downstream from a promoter, such as the trc promoter, andupstream from a cleavage sequence for the protease. If the protease isactive in the expression product, then a band of higher mobility thanthe fusion protein will be observable by Western blotting.

By recovering the band from the gel in which the cleavage is observedand by analyzing the amino terminal sequence thereof by conventionalmethodology, it can be established whether the protein is cleaved at thedesired peptide bond.

The protein which it is desired to produce may be produced, for example,as a fusion protein together with a protein such as glutathioneS-transferase, and the NIa of the present invention can then be used tocleave the linker sequence in vitro. In one alternative, the desiredprotein is directly expressed in a host cell as a fusion proteintogether with said protease and the desired protein is prepared byself-cleavage.

If desired, NIa can easily be produced in a high yield and high purityusing the above methods, and the thus obtained recombinant NIa of thepresent invention can be used as a protease.

If it is desired to chemically synthesize the DNA's of the presentinvention, then these can be prepared by conventional methodology, suchas the phosphite triester method [Hunkapiller, M. et al. (1984), Nature310: 105-111] or by the chemical synthesis of nucleic acids [Grantham,R. et al. (1981), Nucleic Acids Res. 9: r43-r74]. If desired, partialmodification of these nucleotide sequences can be effected byconventional methods, such as by site-specific mutagenesis, using aprimer comprising a synthetic oligonucleotide encoding the desiredmodification [Mark, D. F. et al. (1984), Proc. Natl. Acad. Sci. USA. 81:5662-5666].

Hybridization, as mentioned above, can be established, for example, byusing a probe labeled with [α-³²P]dCTP, for example, in the randomprimer method [Feinberg, A. P. and Vogelstein, B. (1983) Anal. Biochem.132: 6-13], or by nick translation [Maniatis, T. et al. (1982), in“Molecular Cloning A Laboratory Manual” Cold Spring Harbor Laboratory,N.Y.]. The DNA is fixed to a solid phase by a conventional method, forexample by adsorbing to a nitrocellulose membrane or a nylon membrane,and then heating or using ultraviolet radiation. The solid phase is thentypically immersed in a prehybridization solution containing 6×SSC, 5%Denhardt's solution and 0.1% SDS and incubated at 55° C. for 4 hours orlonger. Then, the previously prepared probe is added to theprehybridization solution to a final specific activity of 1×10⁶ cpm/ml,and the mixture incubated at 60° C. overnight. Then, the solid phase iswashed five times repeatedly with 6×SSC for 5 minutes at roomtemperature, followed by washing at 57° C. for 20 minutes, andautoradiography is carried out to determine whether the DNA hashybridized or not. It will be appreciated that this is a specificexample, and that other methods are equally possible.

The desired protein may be prepared by either an intracellular directcutting method and an extracellular cutting method.

Intracellular Direct Cutting Method

DNA encoding NIa is connected with DNA encoding the desired protein viaa cleavage sequence, preferably Gln-Gly, Gln-Ser or Gln-Ala. Theresulting DNA encodes a fusion protein and is inserted into a vectorcomprising a promoter and a terminator and is used to obtain expressionin an appropriate host cell. The resulting, expressed fusion proteinself-cleaves through the protease activity, thereby affording a peptidein which Gly, Ser or Ala is attached by a peptide bond at the N-terminusof the protein of interest. A particularly preferred protein forexpression is IL-11.

Where it is desired to cleave any residues from the N-terminus of theresulting protein, then this may be done as described above, usingaminopeptidase P, for example, to leave an N-terminal Pro residue,should this be desired.

Some expression systems already have aminopeptidase P present. However,if aminopeptidase P is not present, and it is desired to cleaveN-terminal amino acid residues in situ, then an expression vector foraminopeptidase P may be introduced into the host cell. Aminopeptidase Pis a known protein, and a gene sequence for the enzyme is readilyavailable to those in the art.

When it is desired to obtain a protein which starts with Pro at theN-terminus, then it is generally advantageous to extend culture durationin order to allow the cellular aminopeptidase P to act.

As described above, the N-terminal Pro residue can be removed by thecatalytic action of proline iminopeptidase (3.4.11.5), endogenouslyexpressed or expressed by an exogenous expression vector (the enzyme isa known protein, and a gene sequence for the enzyme is readily availableto those in the art). Alternatively, the proline residue can be cleavedafter recovery from the expression system, such as where the fusionprotein is secreted from the cell.

Thus, it can be seen that the present invention permits the productionof proteins having any desired N-terminal residue.

Where the sequence of the present invention results in a polypeptidehaving an Ala N-terminus, then this alanine residue can be removed bythe catalytic action of alanine aminopeptidase (3.4.11.14) which, again,is a known enzyme, and wherein the gene sequence is readily available tothose skilled in the art. This technique also allows the production of apolypeptide having a freely chosen N-terminal residue.

The desired protein can then be isolated and purified by well knownmethods.

Extracellular Cutting Method

NIa can be produced by incorporation of suitable DNA into a vector whichis then included in a suitable expression system. The resulting NIaexpressed by the transformed cell can then purified by use of such as anion exchange column, a gel filtration column or a reverse phase column.Alternatively, the NIa may be expressed as a fusion protein with anotherpolypeptide, such as glutathione-S-transferase or maltose-bindingprotein, which can be separated and purified using a glutathione columnor a maltose column, respectively. After cutting the purified productwith enterokinase or Factor Xa, the NIa can be purified and used.

Meanwhile, the protein precursor is prepared which contains the NIarecognition sequence (cleavage sequence). This may be present in a givenplace, or the protein may form part of a fusion protein with, forexample, glutathione-S-transferase or maltose-binding protein, linked byan appropriate linker (cleavage sequence). Reaction of the precursorwith NIa in a suitable buffer solution cleaves the precursor in vitro.If desired, N-terminal amino acid residues can be removed, as describedabove.

As before, the resulting protein can be isolated and purified by knownmethods.

In respect of the second embodiment of the present invention, we believethe naturally occurring reducing peptide has the sequence shown inSequence ID 12, and consists of 526 amino acids, with a valine residueas the N-terminal.

The naturally occurring DNA encoding the peptide has, we believe, thenucleotide sequence 70 to 1647 indicated in sequence ID number 11.

The peptide of the invention is capable of reducing oxidized glutathioneand dichloroindophenol. This is an accurate description of the peptide,but is somewhat cumbersome, so that the peptide of the invention willalso be referred to herein as the KM31-7 peptide or protein.

The KM31-7 protein originates in the body, or is a mutant or variantthereof, so that there are minimal problems with toxicity and/orantigenicity.

In the second embodiment of the present invention, the polypeptidehaving the sequence −23 to 526 of sequence ID 12 is believed to be aprecursor of the KM31-7 protein. As such, the present invention alsoencompasses this precursor, as well as mutants and variants thereof, andpolynucleotide sequences encoding any of these.

The following discussion is essentially with regard to the secondembodiment of the present invention. It will be understood that many ofthe procedures described above in relation to CYVV NIa are alsoappropriate to the isolation, cloning and. expression of DNA encodingthe peptide of the invention. Any differences essentially arise from thefact that CYVV NIa is a plant virus protein, while the peptide of theinvention is a mammalian protein. General steps for obtaining anexpressed specific polypeptide from mammalian cells by geneticrecombination techniques will now be described.

mRNA encoding the KM31-7 peptide can be obtained and reverse-transcribedinto ds-DNA by well known methods. Any appropriate mammalian cells, celllines or tissue can be used as the source of the original mRNA, but weprefer to use the cell line KM-102 derived from human bone marrowstromal cells [Harigaya, K. and Handa, H. (1985), Proc. Natl. Acad. Sci.USA, 82, 3447-3480].

To extract mRNA from mammalian cells, various methods can be used, suchas the guanidine thiocyanate hot phenol method or guanidine thiocyanateguanidine hydrochloric acid method, but the guanidine thiocyanate cesiumchloride method is generally preferable.

Since the majority of mRNA present in the cytoplasm of eukaryotic cellsis known to have a 3′ terminal poly A sequence, purification ofmammalian mRNA can be effected by adsorption onto an oligo(dT) cellulosecolumn, thereby taking advantage of this characteristic. The eluted mRNAcan then be further fractionated by methods such as sucrose densitygradient centrifugation.

Confirmation that the mRNA does indeed encode the desired peptide can beachieved by translating the mRNA in a suitable system, such as theXenopus laevis oocyte system, the rabbit reticulocyte system or thewheat germ system (supra).

Measurement of the reducing activity of the expression product can beperformed as described below.

i) Determination of Dichloroindophenol Reducing Activity

The methodology for this determination is described by Beinert, H. in“Methods in Enzymology” (1962), 5, 546.

A 50 μM dichloroindophenol (Sigma) preparation is made up with 20 mMphosphate buffer and 0.5 M NaCl (pH 7.8). 1 ml of this preparation isthen placed in a cuvette (SARSTEDT, 10×4×45 mm) and the sample is thenadded for measurement. 15 μl of 1 mM NADPH (Boeringer-Mannheim), made upin the same buffer, is added to the cuvette at room temperature to startthe reaction. Reductase activity can then be determined by following thedecrease in absorption of oxidized dichloroindophenol at 600 nm or byfollowing the decrease in absorption of NADPH at 340 nm.

ii) Determination of Oxidized Glutathione Reducing Activity

The methodology for this assay is described by Nakajima, T. et al. in“New Basic Experimental Methods in Biochemistry (6)—Assay Methods UsingBiological Activity”, 3-34.

A preparation of 10 mM oxidized glutathione (Boeringer-Mannheim) is madeup with 20 mM phosphate buffer and 0.5 M NaCl to a pH of 7.8. 15 μl ofthis preparation are placed in a cuvette (10×4×4 mm) after the samplehas first been placed in the cuvette. 15 μl of 1 mM NADPH prepared inthe same buffer is then added to the cuvette at room temperature tostart the reaction. Glutathione reductase activity can then bedetermined by following the decrease in absorption at 340 nm.

Various methods, as described above, can be used to derive ds-DNA frommRNA. These include the S1 nuclease method, the Land method and the O.Joon Yoo method (supra), but we prefer to use the Okayama-Berg method[Okayama H. and Berg, P. (1982), Mol. Cell. Biol. 2, 161-170] in thisinstance.

The thus obtained ds-cDNA can then be incorporated into a cloning vectorand the resulting recombinant plasmid can be introduced into Escherichiacoli as described above.

A strain containing the desired DNA encoding the peptide of theinvention can be selected by various methods, such as those describedabove in relation to selecting transformants containing NIa DNA, withany appropriate modifications of procedure. For example, if PCR is usedto prepare a probe, then suitable template DNA may either be the cDNAdescribed above, or genomic DNA.

In the second embodiment of the present invention, it is possible to usea primary screen to reduce the number of transformant strains to betested. The primary screen is possible, because the peptide of theinvention is related to the cytokines, so that its mRNA shares the AUUUAmotif common to the mRNA of cytokines [Shaw, G. and Kamen, R. (1986),Cell 46, 659-667.] Thus, a synthetic oligonucleotide probe which iscomplementary to the AUUUA motif can be used in the primary screen.Screening by assay of the production of exogenous protein in mammaliancells can then be performed.

DNA encoding the peptide can then be excised from the vector andsequenced in similar fashion to that described above in relation to NIaDNA. Again, as described above, the DNA fragment can then be introducedinto an appropriate vector and used to transform a prokaryotic oreukaryotic host, as desired.

The KM31-7 protein may be used either alone or in combination with oneor more other therapeutic drugs in the prevention and treatment ofconditions caused by, or related to, oxidative stress, or any diseasecaused by activated oxygen. Such conditions include, but are not limitedto, arteriosclerosis, diabetes, ischemic disorders (such as reperfusiondisorders, ischemic heart disease, cerebroischemia and ischemicenteritis), edema, vascular hyperpermeability, inflammation, gastricmucosa disorders, acute pancreatitis, Crohn's disease, ulcerativecolitis, liver disorders, Paraquat's disease, pulmonary emphysema,chemocarcinogenesis, carcinogenic metastasis, adult respiratory distresssyndrome, disseminated intravascular coagulation (DIC), cataracts,premature retinopathy, auto-immune diseases, porphyremia, hemolyticdiseases, Mediterranean anemia, Parkinson's disease, Alzheimer'sdisease, epilepsy, ultraviolet radiation disorders, radioactivedisorders, frostbite and burns.

Pharmaceutical compositions of the second embodiment of the presentinvention comprise a pharmaceutically active amount of the KM31-7peptide and a pharmaceutically acceptable carrier therefor.

The compositions of the present invention may be administered in variousforms, such as by oral administration in the form of tablets, capsules,granules, powders and syrups, or by parenteral administration in theform of injections, infusions and suppositories. Other suitableadministration forms will be apparent to those skilled in the art.

In the event that the peptide of the invention is to be administered asan injection or infusion, then a pyrogen-free preparation of the peptideis made up in a pharmaceutically acceptable aqueous solution suitablefor parenteral administration. The preparation of the polypeptidesolution so as to conform with the requirements of pH, isotonicity andstability is within the technical expertise of those skilled in the art.

Dosage and form of administration can readily be determined by oneskilled in the art, taking into account such criteria as patientcondition, body weight, sex, age, diet, severity of other infections,administration time and other clinically significant factors. Ingeneral, the normal adult oral dose will be in the range of about 0.01mg to about 1000 mg per day. This amount can be administered in a singledose or as several sub-doses over a period of 24 hours. When the peptideis administered parenterally, about 0.01 mg to about 100 mg peradministration can be given by subcutaneous, intramuscular orintravenous injection.

In order to fully characterize the KM31-7 protein, it was important toobtain an antibody that was specific for this protein. Such an antibodywould be useful in assaying the function, quantification, purificationand tissue distribution of the KM31-7 protein.

Accordingly, a hybridoma producing anti-KM31-7 antibody was obtained byinoculating laboratory animals with the polypeptide produced by E. colitransformed by pMAL31-7, preparing a hybridoma of antibody-producingcells together with myeloma cells, followed by screening and cloning thehybridoma. The antibodies produced by the resulting hybridoma werecapable of recognizing the polypeptide obtained from serum-free culturesupernatant of COS-1 cells transformed with pSRα31-7.

Thus, the prevent invention further provides an antibody, preferably amonoclonal antibody, or an equivalent thereof, which specificallyrecognizes KM31-7 protein, or a mutant or variant of KM31-7 protein.

The antibody of the present invention is directed against the KM31-7protein or a mutant or variant thereof. It will be appreciated that theantibody may be polyclonal or monoclonal, but that the monoclonal formis preferred. This is because of the uncertainty generally associatedwith polyclonal antibodies. For consistency of results, whether fortherapy or for purification of KM31-7 protein, for example, it ispreferred to use monoclonal antibodies.

The antibodies of the invention may be prepared from any suitableanimal. However, problems with antigenicity can arise where the antibodyis non-human. In this respect, it is possible to engineer the antibodiesto more closely resemble human antibodies. This may be achieved eitherby chemical or genetic modification by methods well known in the art.

The present invention also envisages anti-idiotypic antibodies, that is,antibodies whose recognition site recognizes the recognition site of theabove antibodies. Such anti-idiotypic antibodies can be prepared byadministering the original antibody to a suitable animal. It will beappreciated that this process can continue, effectively ad infinitum,with each generation corresponding to either the original or theanti-idiotype. However, if the antibodies are not selected for thecorrect recognition after each generation, then specificity mayeffectively be lost. Anti-idiotypic antibodies may be useful, forexample, in assaying free anti-KM31-7 antibody.

The present invention also envisages fragments of the antibodies of theinvention which are capable of recognizing KM31-7 protein, and moleculescarrying the recognition site of such antibodies. Such fragments andmolecules are referred to herein as “equivalents” of the antibodies ofthe invention.

The plasmid pMAL31-7 was used to transform E. coli, and the expressionproduct was purified and used to immunize laboratory animals. Spleencells from the immunized animals were used to prepare a hybridoma byfusion with myeloma cells, and a clone producing anti-M31-7 monoclonalantibodies was obtained in high concentration and with good stability(this clone was named MKM150-2 and deposited at the FermentationResearch Institute of the Agency of Industrial Science and Technology,Japan, under the deposit number FERM BP-5086). A culture of this cloneyields anti-KM31-7 monoclonal antibodies from the culture supernatant.

The resulting anti-KM31-7 monoclonal antibody reacts immunochemicallywith the fusion protein obtained by introducing and expressing pMAL31-7in E. coli. This antibody also reacts immunochemically with KM31-7protein obtained from the culture supernatants of mammalian cellstransformed with cDNA encoding KM31-7.

In order to produce a monoclonal antibody, the procedures outlined belowwill generally have to be followed. These consist of:

(a) purification of the biopolymer to be used as the antigen;

(b) immunization of mice by injection of the antigen, and preparingantibody-producing cells at the appropriate time from the spleen bysampling and assaying blood;

(c) preparation of myeloma cells;

(d) fusing spleen and myeloma cells;

(e) screening to select the hybridoma group that produces the desiredantibodies;

(f) preparing a single clone (cloning);

(g) culturing the hybridoma for large-scale production of monoclonalantibody, or husbanding mice infected with the hybridoma, as the casemay be; and

(h) assaying the physiological activity or properties as a labellingreagent of the resulting monoclonal antibody.

The production of anti-KM31-7 monoclonal antibodies will now bedescribed with reference to the procedures outlined above. It will beappreciated that it is not necessary to follow precisely the followingdescription, and that it is possible to use any suitable procedure. Forexample, it is possible to use antibody-producing cells and myelomasother than spleen cells as well as antibody-producing cells and myelomasof other mammals. The following procedure represents the currentlypreferred method for obtaining anti-KM31-7 monoclonal antibodies.

(a) Antigen Purification

The fusion protein obtained by expressing pMAL31-7 in E. coli andpurifying the product is an effective antigen. A culture of E. coli TB-1transformed with pMAL31-7 was induced with isopropylβ-D-thiogalacto-pyranoside (IPTG). The expressed fusion protein was thenpurified by affinity chromatography using an amylose resin column (NewEngland BioLabs). KM31-7 protein purified from the serum-free culturesupernatant of COS-1 cells transformed with pSRα31-7 is also aneffective antigen.

(b) Preparation of Antibody-Producing Cells

The purified fusion protein obtained in (a) is mixed with Freund'scomplete or incomplete adjuvant, or an adjuvant such as potash alum, andlaboratory animals are then immunized with the resulting vaccine. BALB/cmice are a preferred choice for use as the laboratory animals, becausethe majority of useful myelomas derived from mice are derived fromBALB/c mice. Moreover, the characteristics of these mice have beenstudied in a great amount of detail. Furthermore, if both theantibody-producing cells and the myeloma are from BALB/c mice, then theresulting hybridoma can be grown in the abdominal cavity of BALB/c mice.Thus, the use of BALB/c mice offers the advantage of enabling monoclonalantibodies to be easily obtained from ascites without having to employcomplex procedures. Nevertheless, the present invention is not limitedto the use of BALB/c mice.

The antigen may be administered in any suitable form, such as bysubcutaneous injection, intraperitoneal injection, intravenousinjection, intracutaneous injection or intramuscular injection, and weprefer subcutaneous injection or intraperitoneal injection.

Immunization may be performed once or on a plurality of occasions withsuitable intervals. The preferred regimen is to immunize and then boost,one or more times, at intervals of from 1 to 5 weeks. The effectivenessof later procedures can be improved if the antibody titer to saidantigen in the serum of the immunized animals is regularly assayed, andanimals having a sufficiently high antibody titer are used to provideantibody-producing cells. Antibody-producing cells for subsequent fusionare preferably isolated from an animal 3 to 5 days after the finalimmunization.

Methods for assaying antibody titer include, for example, various knowntechniques, such as radioisotope immunoassay (RIA), enzyme-linkedimmunosorbent assay (ELISA), the fluorescent antibody technique andpassive hemagglutination, but RIA and ELISA are preferable for theirsensitivity, speed, accuracy and potential for automation.

A suitable form of ELISA is as follows. Antigen is adsorbed onto a solidphase and then the solid phase surface is exposed to a protein unrelatedto the antigen, such as bovine serum albumin (BSA), to block any areasof the surface which have no adsorbed antigen. The solid phase is thenwashed, after which it is exposed to a serially diluted sample of theprimary antibody (e.g. mouse serum). Any anti-KM31-7 antibody in thesample binds to the antigen. After washing, secondary, enzyme-linked,anti-mouse-antibody is added and is allowed to bind to bound mouseantibody. After washing, enzyme substrate is added and the antibodytiter can then be calculated by measuring a parameter, such as colorchange, caused by decomposition of the substrate.

(c) Myeloma Cell Preparation

Established mouse cell lines are preferably used as the source ofmyeloma cells. Suitable examples of such cell lines include myeloma cellline P3-X63 Ag8-U1 (P3-U1) from 8-azaguanine resistant mice of BALB/corigin [Current Topics in Microbiology and Immunology, 81, 1-7 (1978)],P3-NSI/1-Ag4.1 (NS-1) [European J. Immunology, 6, 511-519 (1976)],SP2/O-Ag14 (SP-2) [Nature, 276, 269-270 (1978)], P3-X63-Ag8.653 (653)[J. Immunology, 123, 1548-1550 (1979)] and P3-X63-Ag8 (X63) [Nature 256,495-497 (1975)]. These cell lines may be subcultured in suitable media,such as 8-azaguanine medium (RPMI-1640 medium containing 8-azaguanine,1.5 mM glutamine, 5×10⁻⁵ M 2-mercaptoethanol, 10 μg/ml gentamycin and10% fetal calf serum), Iscove's Modified Dulbecco's Medium (IMDM) orDulbecco's Modified Eagle's Medium (DMEM). The cell count is elevated toat least 2×10⁷, on the day of fusion, by subculturing with normalmedium, also known as complete GIT [5.5 ml of MEM non-essential aminoacids solution (NEAA, Gibco), 27.5 ml of NCTC109 (Gibco), 6 ml ofpenicillin-streptomycin solution (Sigma) and 11 ml of glutamine 200 mMsolution (Sigma) in 500 ml of GIT medium (Wako Pure Chemical Industry)],3 to 4 days before cell fusion.

(d) Cell Fusion

The antibody-producing cells are plasma cells and their precursorlymphocytes. These may be obtained from any appropriate site of anindividual animal, such as the spleen, lymph nodes, peripheral blood orany suitable combination of these. However, spleen cells are mostcommonly used.

Antibody-producing cells are harvested, 3 to 5 days after the finalimmunization, from mice having at least the prescribed antibody titer.The resulting antibody-producing cells are then fused with the myelomacells obtained in (c) above. The process most commonly used at presentis to fuse the spleen cells with the myeloma cells using polyethyleneglycol, owing to the relatively low level of cellular toxicity and easeof manipulation of this compound. This process is performed as follows.

Spleen cells and myeloma cells are thoroughly washed with medium orphosphate buffered saline (PBS), mixed so that the ratio of spleen cellsto myeloma cells becomes roughly between 5 and 10:1, and then subjectedto centrifugal separation. The supernatant is discarded and the clump ofcells is thoroughly broken up, and then a mixed solution of polyethyleneglycol (PEG, molecular weight: 1000 to 4000) is added with stirring.After several minutes, the cells are subjected to centrifugalseparation. The supernatant is again discarded, and the settled cellsare suspended in a suitable amount of complete GIT containing 5 to 10ng/ml of mouse IL-6 and then transferred to the wells of a cultureplate. Once cell growth has been confirmed in each well, the medium isreplaced with HAT medium (complete GIT containing 5 to 10 ng/ml of mouseIL-6, 10⁻⁶ to 10⁻³ M hypoxanthine, 10⁻⁸ to 10⁻⁷M aminopterin and 10⁻⁶ to10⁻⁴ M thymidine).

(e) Selection of Hybridoma Groups

The culture plate is incubated in a CO₂ incubator at 35 to 40° C. for 10to 14 days. During this time, fresh HAT medium equivalent to half theamount of medium is added every 1 to 3 days.

The myeloma cells are from an 8-azaguanine-resistant cell line and boththe myeloma cells and hybridomas consisting only of myeloma cells cannotsurvive in HAT medium. However, hybridomas comprising anantibody-producing cell part, including hybridomas of antibody-producingcells and myeloma cells, can survive. Hybridomas consisting only ofantibody-producing cells are mortal, so that hybridomas consisting ofboth myeloma and antibody-producing cells can be selected by culturingin HAT medium.

HAT medium is replaced with HT medium (wherein aminopterin has beenomitted from HAT medium) in those wells in which hybridomas which havebeen observed to develop colonies. A portion of the culture supernatantis then removed and anti-KM31-7 antibody titer is assayed by, forexample, ELISA.

The above process is described with respect to an 8-azaguanine-resistantcell line, but other cell lines can also be used, provided that theypermit the selection of hybridomas. The composition of the medium usednaturally also changes in such cases.

(f) Cloning

Hybridomas from (e) which have been determined to produce anti-KM31-7specific antibody are transferred to a different plate for cloning.Various cloning methods can be used, such as the seeding method (whereinthe hybridomas are subjected to limiting dilution analysis so that eachwell contains only one hybridoma), the soft agar method (wherein seededcolonies are taken in soft agar medium), the seeding method (wherein asingle cell is removed by a micro-manipulator), and the sorter cloningmethod (wherein individual cells are separated by a cell sorter).Limiting dilution analysis is used most frequently due to itssimplicity.

Cloning, such as by limiting dilution analysis, is repeated 2 to 4 timesfor those wells in which an antibody titer continues to be observed. Aclone consistently exhibiting the production of anti-KM31-7 antibody isselected as the hybridoma of choice.

(g) Preparation of Monoclonal Antibody by Hybridoma Culture

The hybridoma of choice from (f) is then cultured in ordinary medium.Large-volume culture is performed by rotary culturing using either alarge culture bottle or a spinner. Anti-KM31-7 monoclonal antibodies canbe obtained by subjecting the cell supernatant to gel filtration, andthen collecting and purifying the IgG fraction. In addition, thehybridoma can also be grown in the abdominal cavity of the same strainof mouse (e.g. the above-mentioned BALB/c mice) or Nu/Nu mice, forexample. A simple method for performing this step is to use a monoclonalantibody preparation kit (e.g., MAbTrap GII of Pharmacia).

(h) Identification of Monoclonal Antibody

Determination of the isotype and subclass of the monoclonal antibodyobtained in (g) can be performed as described below. Examples ofidentification techniques which can be used include the Ouchterlonymethod, ELISA and RIA. Although the Ouchterlony method is simple, thereis a drawback, in that the monoclonal antibody must be concentrated incases in where concentration is excessively low.

If either ELISA or RIA is used, the culture supernatant can be directlyexposed to a solid phase on which antigen has been adsorbed. Secondaryantibodies specific for each type of IgG subclass can then be used toidentify subclass type. In the alternative, an isotyping kit (forexample, the mouse monoclonal antibody isotyping kit of Amersham) can beused. Protein quantification can be performed by the Folin-Lowry methodor by measuring absorbance at 280 nm [1.4 (OD₂₈₀)=1 mg/ml ofimmunoglobulin].

In the accompanying Examples, the monoclonal antibody obtained from thehybridoma designated MKM150-2 was determined to be of the IgG classisotype and was identified as belonging to the IgGl subclass.

The monoclonal antibody obtained in accordance with the presentinvention has a high-specificity for KM31-7 protein. Moreover, sincemonoclonal antibody is obtained consistently and in good quantities byculturing the above-mentioned hybridoma, it can be used for theisolation and purification of KM31-7 protein, and the isolation andpurification of KM31-7 protein with said monoclonal antibody by immuneprecipitation using an antigen-antibody reaction forms a part of thepresent invention.

The present invention will now be illustrated with reference to theaccompanying, non-limiting Examples. All solutions are aqueous andprepared from deionized water, and those expressed in the form ofpercentages are w/v, unless otherwise specified. If methods are notspecified, then they can be found in “Molecular Cloning—A LaboratoryManual” [second edition, Sambrook, J., Fritsch, E. F. and Maniatis, T.(1989), Cold Spring Harbor Laboratory Press]. Methods of preparation ofsolutions and other media are given in the later section entitled“Media” where they are not given in the Examples. Methods referred to inthe Examples without techniques should be construed in the context ofpreceding Examples.

A) Source Material for CYVV

A leaf infected with the clover yellow vein virus and which had beenstored in the frozen state [CYVV-isolate No. 30: Uyeda, I. et al.(1975), Ann. Phytopath. Soc. Japan 41: 192-2031 was milled with 10volumes of inoculation buffer.

Propagation of CYVV was carried out using Vicia faba cultivarWase-Soramame which had developed a second adult leaf. An adult leaf wassprayed with carborundum (400 mesh) and was then inoculated with theliquid obtained above using a glass spatula. About eight to ten daysafter the inoculation, the inoculated leaf had developed a mesh-likemosaic condition. This leaf was removed and used as a source of CYVV.

B) Purification of Virus

Leaves isolated in A) above were chopped with a mincer containing 3volumes of extraction buffer. After chopping, the leaves were furtherground in a mortar and pestle. The resulting preparation was stirredslowly at room temperature for 1 hour using a motor equipped with astainless steel agitating blade. The liquid preparation was thensqueezed out through a double layer of gauze.

All of the subsequent procedures were carried out at 4° C.

The crude liquid was mixed with a half volume of chloroform and themixture was blended in a Waring blender, after which the preparation wascentrifuged at 6,000×g for 15 minutes. Following centrifugation, theaqueous layer was collected, and polyethylene glycol (PEG #6,000) wasadded to this fraction to a final concentration of 4% v/v.

The resulting mixture was stirred over ice for 1 hour and allowed tostand on ice for another 1 hour. The thus obtained liquid was subjectedto centrifugation at 6,000×g for 15 minutes, and the precipitate wasrecovered as the virus-containing fraction. This precipitate wassuspended in 50 ml of a 10 mM phosphate buffer (pH 7.4) containing 0.5 Murea and then mixed with an equal volume of carbon tetrachloride, andthe resulting preparation was stirred vigorously for 5 minutes, afterwhich time the preparation was subjected to centrifugation at 3,000×gfor 10 minutes. The aqueous phase was then ultracentrifuged at 4° C. at30,000 rpm for 90 minutes using an Hitachi RP-30 rotor. The resultingpellet was recovered as the virus-containing fraction.

The pellet was suspended in 10 mM phosphate buffer containing 1% TritonX100 (pH 7.4) and this suspension was subjected to centrifugation at 4°C. at 8,000×g for 1 minute. The resulting supernatant was layered on agraduated 10 to 40% sucrose density gradient column tube (40%: 10 ml,30%: 10 ml, 20%: 10 ml, 10%: 10 ml; and which had been allowed to standovernight at 4° C.) which had previously been prepared with 10 nMphosphate buffer (pH 7.4). Once the tube had been layered with thesupernatant, it was subjected to ultracentrifugation at 4° C. at 23,000rpm for 120 minutes in a Hitachi PRS25 rotor.

After centrifugation, the sucrose density gradient column tube wasfractionated using a fractionater (Model UA-2: manufactured by ISCO Co.)equipped with an OD_(260 nm) detector. The fractions having a sucrosedensity of about 20 to 30% and absorbing at OD_(260 nm) were taken asthe virus-containing fraction.

The recovered virus fraction was diluted 2-fold with 10 mM phosphatebuffer (pH 7.4) and subjected to centrifugation at 4° C. at 40,000 rpmfor 90 minutes using a Hitachi RP-65 rotor. The resulting precipitatewas resuspended in 10 mM phosphate buffer (pH 7.4) to yield a purifiedvirus solution.

C) Isolation of Viral RNA

The genomic RNA of CYVV comprises about 10,000 bases and ispolyadenylated at its 3′-terminus. The combination of length andpolyadenylation make it difficult to extract the full-length RNA withoutany loss when using either the conventional phenol/sodium dodecylsulfate method or the guanidinium thiocyanate method. Accordingly, theviral genomic RNA was prepared by the alkaline sucrose density gradientcentrifugation method.

500 μl of the virus solution (which we had calculated to contain 2 mg ofthe virus on the basis that 1 mg/ml of virus has an OD_(260nm) of 2.5)was added to 500 μl of degradation solution and the mixture was allowedto stand at room temperature for 20 minutes. After this time, themixture was layered over a 0% to 33.4% sucrose density gradient columntube (33.4%: 1.4 ml, 30.4%: 7.6 ml, 27%: 7.0 ml, 23%: 6.3 ml, 18.7%: 5ml, 12%: 3.2 ml, 0%: 2.7 ml; and which had been allowed to stand at 4°C. overnight) which had been prepared with 1×SSC. The buffer used wasalso 1×SSC, and the layered tube was then subjected toultracentrifugation in a Hitachi RPS-27 rotor at 15° C. at 24,000 rpmfor 9 hours. After this time, the bottom of the tube was punctured inorder to fractionate the sucrose density gradient. The viral genomic RNAfraction was identified by measuring the absorbance of each fraction at260 nm. Sedimentation of the viral genomic RNA occurred at a sucroseconcentration of about 20 to 30%. Genomic RNA was obtained in a yield ofabout 25 μg.

D) Synthesis of Viral cDNA

cDNA was synthesized using the genomic RNA prepared in C) above as atemplate. cDNA synthesis was carried out using the cDNA Synthesis SystemPlus (manufactured by Amersham). The resulting cDNA was purified on aSephadex G50 column (registered trademark, manufactured by Pharmacia),and a poly C chain was added to the 5′ terminus of the purified cDNAusing dCTP and terminal deoxynucleotide transferase (manufactured byBethesda Research Laboratories). A preparation of plasmid pBR322(manufactured by Bethesda Research Laboratories) was made by digestingthe plasmid with the restriction enzyme PstI and then adding a 3′ poly Gchain at both termini. The cDNA was then added to this preparation, andthe mixture was incubated at 65° C. for 5 minutes, after which time itwas incubated at 57° C. for 2 hours. The resulting mixture was thengradually cooled to allow annealing of the poly C chain of the cDNA withthe poly G chain of the plasmid.

E) Transformation

Escherichia coli strain HB 101 was transformed with the novel plasmidprepared in D) above by the calcium chloride method. A seed culture ofE. coli strain HB 101 was prepared by shaking overnight in liquid LBmedium. 0.5 ml of this seed culture was used to inoculate 50 ml of freshliquid LB medium and the inoculum was cultured with shaking at 37° C.until an OD_(550 nm) of 0.5 was obtained. Bacterial cells were recoveredby centrifugation at 4° C. at 5,000×g for 5 minutes and the resultingpellet was gently suspended in 25 ml of Tris-calcium buffer and allowedto stand on ice for 5 minutes. The resulting suspension was centrifugedagain at 4,000×g for 4 minutes and the pellet was suspended in 5 ml ofTris-calcium buffer and allowed to stand on ice for 2 hours to yieldcompetent cells.

100 μl of the novel plasmid obtained in D) above were added to 200 μl ofthe competent cells obtained above, and the mixture was allowed to standon ice for 30 minutes. After this time, the mixture was incubated at 42°C. for 2 minutes and then 1 ml of liquid LB medium was added, and theresulting mixture was cultured with shaking at 37° C. for a further onehour. The resulting mixture was spread onto solid LB medium containing12.5 μg/ml tetracycline hydrochloride and 1.5% w/w agar. The cells werecultured at 37° C. overnight to provide a cDNA clone library.

F) Preparation and Selection of Plasmid pNS51

The cDNA recombinant plasmid library was analyzed using the alkaline-SDSmethod. In more detail, the method was as follows.

2 ml of liquid LB medium was inoculated with a colony of bacteriaresistant to tetracycline but sensitive to ampicillin, obtained from thesolid LB culture of E) above, and the resulting suspension was culturedwith shaking at 37° C. overnight. Cells were recovered by centrifugationat 10,000×g and suspended in 70 μl of lysis buffer containing a further16 μl of lysozyme solution (10 mg/ml). After agitating for 5 seconds tocreate a suspension, the suspension was allowed to stand at roomtemperature for 5 minutes. After this time, 160 μl of alkaline-SDSsolution were added to the suspension and the resulting mixture wasfirst mixed by inverting the tube several times and then allowed tostand on ice for 5 minutes. 120 μl of 5 M potassium acetate were nextadded to the mixture which was then again allowed to stand on ice for 5minutes. After this time, the mixture was centrifuged at 10,000×g for 5minutes at 4° C.

After centrifugation, the supernatant was transferred to a fresh tube,and 250 μl of isopropanol was further added to the tube and theresulting mixture was allowed to stand on ice for 30 minutes. After thistime, the mixture was again centrifuged at 10,000×g for 5 minutes, andthe resulting pellet was dissolved in 100 μl of TE buffer [10 mM Tris, 1mM EDTA (pH 8.0)]. Phenol extraction was then performed by adding anequal amount of phenol/chloroform/isoamyl alcohol (25:24:1) to theresulting mixture with vigorous mixing, and centrifuging the mixture at10,000×g for 5 minutes at 4° C. (hereinafter, this procedure is referredto as phenol extraction).

100 μl of the aqueous layer obtained from the centrifugation was mixedwith 10 μl of 3 M sodium acetate (pH 5.2) and 250 μl of ethanol, and theresulting mixture was allowed to stand on dry ice for 10 minutes,followed by centrifugation at 10,000×g for 5 minutes to recover thenucleic acid fraction (hereinafter, this procedure, using the samerelative volumes of supernatant, ethanol and sodium acetate solution, isreferred to as ethanol precipitation).

The resulting pellet was suspended in 50 μl of a solution of RNase A (10μg/ml in TE buffer) and then incubated at 37° C. for 1 hour. After thistime, 30 μl of a solution of polyethylene glycol (2.5 M sodium chloride,20% polyethylene glycol #8,000) was added to the incubated suspension,and the mixture was allowed to stand on ice for 1 hour. After this time,the mixture was centrifuged at 10,000×g for 5 minutes at 4° C. torecover the DNA fraction as a pellet, and ethanol precipitation wasperformed twice more to yield purified plasmid DNA.

The purified plasmid DNA was cleaved with restriction enzyme PstI andsubjected to 1 agarose gel electrophoresis using TBE solution. Afterelectrophoresis, the gel was subjected to Southern blot hybridization(Southern, E. M. (1975), J. Mol. Biol. 89: 503-517]. More specifically,the gel was shaken in denaturation solution for 40 minutes, thentransferred to and shaken in neutralization buffer for 2 hours.

After shaking, the gel was transferred onto a polyurethane ester spongecontaining 20×SSC to transfer the DNA to a piece of Hybond-N membrane(registered trademark of Amersham) situated on the sponge. After the DNAhad been allowed to transfer to the Hybond-N membrane, the membrane wasshaken in 1×SSC for 10 minutes and then dried at 80° C. for 1 hour tofix the DNA. The membrane was then placed in prehybridization solution[5 ml of formamide, 1 ml of 50× Denhardt's solution, 2.5 ml of 20×SSC,100 μl of yeast tRNA (50 mg/ml), 100 μl of 10% SDS and 1.3 ml ofredistilled water] and incubated at 50° C. for 3 hours.

Viral genomic RNA which had been cleaved with Mg²⁺ was used as a probe.More particularly, 4 μl of 5 μl denaturation buffer was added to 16 μlof the solution obtained in C) above containing 1 μg viral RNA, and themixture was incubated at 37° C. for 3 hours. 5 μl of denatured RNAsolution containing 100 ng of RNA (calculated on the basis that 1 mg/mlof RNA has an OD₂₆₀ of 20) was recovered by ethanol precipitationfollowed by heating at 70° C. for 5 minutes and then quenching on ice.

4 μl of 5× labelling buffer, 1 μl of 3.3 mM [γ-³²P] ATP (0.37 MBq), and10 μl of redistilled water were added to the denatured RNA solution. 20U of T4 polynucleotide kinase solution [manufactured by Takara Shuzo]were then added to the mixture, followed by incubation at 37° C. for 30minutes. Unincorporated [γ-³²P] ATP was removed by repeating ethanolprecipitation five times.

The resulting labelled probe was added to hybridization solution [5 mlof formamide, 2 ml of 50% dextran sulfate, 200 μl of 50× Denhardt'ssolution, 2.5 ml of 20×SSC, 50 μl of yeast tRNA (50 mg/ml), 100 μl of10% SDS, 1.3 ml of redistilled water] to a final concentration of 5×10⁵cpm/ml, and the Hybond-N membrane obtained above was transferred to thissolution and subjected to hybridization by incubation at 50° C.overnight, followed by washing with shaking at 6° C. with 2×SSCcontaining 0.1% sodium dodecyl sulfate (SDS). This procedure wasrepeated three times, and the Hybond-N membrane was then further washedwith 0.1×SSC containing 0.1% SDS with shaking at room temperature for 1hour, and then dried.

Autoradiography revealed a plasmid which we designated pNS51 which hadan insertion fragment comprising about 6,500 base pairs (bp) which hadhybridized with the viral genomic RNA.

G) Sequence Determination of pNS51 Insert

In order to prepare a restriction map of the cDNA cloned in pNS51, theplasmid was digested with each of the restriction enzymes; BamHI, EcoRI,Hind III, KpnI, NcoI, PstI, SalI, SphI, SacI, SmaI, XhoI, XbaI, andpairs thereof. The resulting map is shown in FIG. 1.

Subsequently, each of the fragments obtained by digestion with therestriction enzymes SalI and PstI was inserted into M13 mp19 RF DNA.This was done by digesting pNS51 with the restriction enzymes PstI andSalI and running the cleavage products on a 5% polyacrylamideelectrophoresis gel using 1× TBE. After electrophoresis, the gel wasstained with 1 μg/ml of ethidium bromide in order to be able to identifythe bands under UV light. Strips of the gel containing each DNA bandwere excised with a razor and subjected to electroelution with anelectroeluter (manufactured by Amicon) equipped with Centricon-30(manufactured by Amicon).

Each fragment was then inserted, using a DNA ligation kit [manufacturedby Takara Shuzo], into M13 mp19 which had previously been digestedeither with SalI only, or with both of PstI and SalI, and which had thenbeen treated with alkaline phosphatase.

The resulting recombinant M13 mp19 RF-DNA (wherein the fragments hadbeen inserted using T4 DNA ligase) was introduced into E. coli strainJM109 using the rubidium chloride method. A single colony of strainJM109 which had been cultured on M9 minimum agar medium was inoculatedinto and cultured in liquid SOB medium with shaking overnight. 0.6 ml ofthis overnight culture was inoculated into 50 ml of fresh liquid SOBmedium and cultured with shaking at 37° C. until the OD_(600 nm) reached0.5. The cells were then recovered by centrifugation at 5,000×g at 4° C.for 10 minutes and the pellet was gently suspended in 25 ml of TFB1buffer and allowed to stand on ice for 20 minutes.

The suspension was again centrifuged at 3,000×g for 5 minutes, and thepellet was suspended in 2 ml of TFB2 buffer and then allowed to stand onice for 20 minutes to provide competent cells.

The recombinant M13 mp19 RF-DNA obtained above was used in amounts ofbetween 1 and 10 μl after ligation and mixed with 100 μl of thecompetent cells and allowed to stand on ice for 1 hour. After this time,the mixture was incubated at 42° C. for 1.5 minutes and then 500 μl ofliquid SOC medium was added to the mixture which was cultured withshaking at 37° C. for a further 1 hour.

2× YT medium containing 0.8% agar, 30 μl of 100 mM IPTG, 10 μl of 10%5-bromo-4-chloro-3-indolyl-↓-galactoside (X-gal) and 100 μl of anindicator bacteria (an overnight culture of strain JM109 cultured withshaking in 2× YT medium at 37° C.) were added to the resulting culturewith shaking, and the mixture was poured onto solid 2× YT mediumcontaining 1.2% agar. After the mixture had solidified, it was culturedat 37° C. overnight. Recombinant phages subsequently showed as whiteplaques.

Each of the resulting white plaques was inoculated into liquid 2× YTmedium containing the indicator bacteria in an amount of 1/100, and thiswas cultured with shaking at 37° C. overnight. After centrifugation at10,000×g for 1 minute, the supernatant was frozen or stored at 4° C. asa phage solution, and the cell pellet was processed in a standardprocedure for preparing a plasmid to recover RF double-stranded DNA(hereinafter, abbreviated as RF-DNA), which is a replicationintermediate of the phage. The presence of an insertion fragment wasconfirmed by digestion with a restriction enzyme.

Thus, 51SS/M13 mp19 (i.e. M13 mp19 containing a SalI/SalI fragmentwhich, in turn, is a portion of 51PS5′/M13 mp19 genome containing aPstI-SalI fragment from the 5′ region of the CYVV genome) was obtained.RF-DNA's were purified from these phage clones by alkaline-SDS andcesium chloride density gradient centrifugation in accordance withstandard procedures.

5 μg of the resulting purified DNA was first cleaved with BamHI toobtain a 5′ sticky end, and then cleaved further with KpnI to obtain a3′ sticky end. Next, in accordance with the protocol accompanying theErase-a-base system (manufactured by Promega), exonuclease was added forthe stepwise deletion of bases from the 5′ sticky end of each cDNAlinearized plasmid. Treatment with exonuclease was continued for 10minutes, and samples were removed at different times between 1 and 10minutes. Since the vector has a 3′ sticky end, it is not attacked by theexonuclease. The various samples obtained over the period of 1 to 10minutes were then blunt-ended by treatment with S1 nuclease and ligatedinto a circular form using T4 DNA ligase.

The resulting, circular, recombinant M13 mp19 RF-DNA was introduced intoE. coli strain JM 109 using the rubidium chloride method describedabove. Accordingly, recombinant phages having variable length cDNAinserts were obtained as a white plaques.

RF-DNA was extracted from the recombinant phage subclones obtained aboveby conventional means to select clones having inserts of differentlength. The selected clones were separately cultured by inoculation intoliquid 2× YT medium containing an indicator strain of E. coli and werecultured with shaking at 37° C. overnight. After this time, 1.5 ml ofeach culture was centrifuged at 4° C. at 10,000×g for 5 minutes. One mlof the supernatant was transferred to a fresh tube and 250 μl of a 20%polyethylene glycol aqueous solution (20% polyethylene glycol #8000, 2.5M sodium chloride) was then added to the tube with mixing and thenallowed to stand on ice for 30 minutes. The mixture was subsequentlycentrifuged at 10,000×g for 5 minutes at 4° C., and the resulting pelletwas suspended in 100 μl of TE buffer. Single stranded DNA (hereinafterabbreviated as ssDNA) phage genomes were then recovered from eachpreparation by phenol extraction and ethanol precipitation as before.

Nucleotide sequencing of each of the subclones obtained above was thenperformed by the dideoxy chain termination procedure, using the ssDNA asa template. More specifically, the phage ssDNA was labeled with[α-³²P]dCTP (220 TBq/mmol, manufactured by Amersham) using the7-deaza-sequenase version 2.0 dCTP kit (manufactured by USB). Theresulting reaction product was run on a 5% polyacrylamideelectrophoresis gel containing 8 M urea and using 1× TBE as the buffer.The gel was dried, and the nucleotide sequence was then determined byautoradiography.

By analyzing the nucleotide sequences of the 5′-PstI-SalI fragment andthe M-SalI—SalI fragment of pNS51, we were able to determine a total of3,839 bases.

As a result of the search for an open reading frame (hereinafterabbreviated as ORF) in the sequence obtained above, a single ORF for apolypeptide was detected on the + sense strand of the viral genome. Thepolypeptide sequence encoded in the ORF was then determined. A homologysearch comparing this polypeptide sequence with known sequences usingGeneBank revealed homology with TEV-HAT.

It is known that viruses such as TEV, plum pox virus or poliovirus (ananimal virus) mature through the action of a protease, and that theprotease generally cleaves a peptide bond containing a Gln-Ala, Gln-Seror Gln-Gly link [Willink J. and van Kammen, A. (1988), Arch. Virol. 98:1-26]. We were also able to detect three cleavage sequences in thededuced polypeptide sequence, as a result of analysis of the cleavagesite(s) of the coat protein of CYVV-No. 30 [Uyeda, I. et al. (1991),Intervirology 32: 234-245]. From one of these cleavage sites, apolypeptide of from Gly at position 4 to Gln at position 437 of theamino acid sequence was determined to be Nuclear Inclusion a from CYVV.

H) Preparation of a CYVV-NIa/IL-11 Fusion Protein

The DNA identified in G) above as coding for CYVV NIa was tandemlylinked, in-frame, at its 3′ end to DNA encoding human interleukin 11(IL-11), via a linking sequence encoding Gln-Ala.

The following were examined:

first, whether or not the DNA identified in G) above encodes an NIahaving proteolytic activity;

second, whether or not the protease cuts the desired amino acidsequence;

third, whether or not the protease activity of the NIa is stillexhibited when the NIa is part of a fusion protein together with adesired heterologous protein in a manner similar to that existing in avirus-infected cell; and

fourth, whether or not any heterologous protein obtained through thecleavage of such a fusion protein maintains integrity, structure andfunction.

Since the NIa itself is produced by excision from a viral precursorpolypeptide, the NIa cistron has no initiation codon. Therefore, it isnecessary to add an initiation codon, ATG, to the 5′ terminus of the NIagene. The 3′ terminus of the NIa gene must also be connected with the 5′terminus of the IL-11 gene within the same ORF. In order to solve theseproblems, the NIa was modified using the polymerase chain reaction (PCR)technique by utilizing a XhoI cleavage site present in the center of theNIa gene.

In order to add the initiation codon ATG and a recognition site for therestriction enzyme NcoI suitable for cloning to the 5′ terminus of NIa(NIa5′), PCR primers were prepared. The sequences prepared were5′GTCCATGGGGAAAAGTAAGAGAACA3′ (referred to as NSATG; sequence ID number:3) and 5′ACTCTGAGACCGTGCTCGAG3′ (referred to as NSX1; sequence IDnumber: 4).

0.8 μl of a dNTP solution (25 mM each of dATP, dTTP, dCTP and dGTP), 10×Taq buffer (manufactured by Promega) and 1 μg of each of the resultingprimers were added to 1 μg of plasmid pNS51 DNA, and the mixture wasmade up to 100 μl with redistilled water. 5 U of DNA polymerase made upin 10× Taq buffer were added to effect PCR. For the PCR program, a cycleof 92° C. for 1 minute, 37° C. for 1 minute and 72° C. for 2 minutes wasrepeated 20 times followed by a single cycle of 92° C. for 1 minute, 37°C. for 1 minute and 72° C. for 30 minutes.

The resulting amplified DNA was subjected to phenol extraction andethanol precipitation, and then run on a 5% polyacrylamideelectrophoresis gel. One band containing DNA was detected by ethidiumbromide staining, and this band was excised from the gel and subjectedto electroelution with a Centreluter (manufactured by Amicon) equippedwith Centricon-30 (manufactured by Amicon). After the elution, theeluted DNA fragment was concentrated and recovered by centrifugation at7,500×g using Centricon (Amicon) at 4° C. for 45 minutes. The resultingDNA concentrate was further purified by phenol extraction and ethanolprecipitation.

Separately, 1 μg of plasmid pKK388-1 (manufactured by Clonetech) inwhich a SacI recognition site had been replaced with a recognition sitefor XhoI, was cleaved with the restriction enzymes NcoI and XhoI anddephosphorylated with Calf Intestine Alkaline Phosphatase [hereinafterabbreviated as CIAP; manufactured by Takara Shuzo]. The resulting DNAwas ligated by means of a ligation kit (manufactured by Takara Shuzo)with 100 ng of pKK388-1 which had also been cleaved with NcoI and XhoIand dephosphorylated as above, and the recombinant DNA thus obtained wascloned into E. coli JM 109. Plasmid pKNI5′ which had an insertion in thenormal orientation downstream from the trc promoter of pKK388-1 wasthereby obtained (FIG. 2).

Plasmid pCD20-2 [Kawashima, I. et al. (1991), FEBS L. 283: 199-202]contains cDNA coding for an IL-11 precursor (Pre-IL-11) and having asecretion signal sequence. This plasmid was cleaved with the restrictionenzymes BamHI and ApaI, and a region having both the IL-11 precursor(Pre-IL-11) and SV40 promoter was excised. The fragment was ligated intothe BamHI and ApaI sites of pBLUESCRIPT II SK+ and then again cleavedwith the restriction enzymes XhoI and KpnI, resulting in a gene whichcodes for a protein devoid of the N-terminus of mature IL-11(Mat-IL-11).

The resulting Mat-IL-11 fragment (devoid of its N-terminus) wasintegrated using T4 ligase into pKNI5′ which had previously been cleavedwith the restriction enzymes XhoI and KpnI and also treated with CIAP.We designated the resulting construct pKNI5IL (FIG. 3). In order toligate the specific sequence at the C-terminus of NIa with Mat-IL-11 viaAla while keeping the same reading frame, four kinds of PCR primers weresynthesized:

5′ AGGAAAAGAGTTCCTCGAGC 3′, (referred to as NSX2; sequence ID number: 5)5′ AATTGTTCATTCCAAGCACCTGGGCCACCACCTGGC 3′, (referred to as NSJ001P;sequence ID number: 6) 5′ GCCAGGTGGTGGCCCAGGTGCTTGGAATGAACAATT 3′,(referred to as NSJ002N; sequence ID number: 7) and5′ TTGTCAGCACACCTGGGAGCTGTAGAGCTC3′. (referred to as ILSAC; sequence IDnumber: 8)

The first PCR reaction carried out used the pair of primers NSX2 andNSJ002N, with pNS51 DNA as template to amplify a region of the insertcoding for the C-terminus of NIa (designated the CNI3 region).Separately, another PCR reaction was performed using the pair of primersNSJ001P and ILSAC, with pNS51 DNA as template to amplify a region of theinsert coding for N-terminus of the IL-11 peptide (designated the 5′ ILregion). For the PCR program, a cycle of 92° C. for 1 minute, 37° C. for1 minute and 72° C. for 2 minutes was repeated 20 times followed by asingle cycle of 92° C. for 1 minute, 37° C. for 1 minute and 72° C. for30 minutes. The products obtained from each PCR procedure were extractedwith phenol and precipitated with ethanol, and then subjected to 5%polyacrylamide gel electrophoresis. A gel band containing the amplifiedDNA band with the gel was excised and the DNA was electrically recoveredfrom the gel slice using the electroelution method described above. Theresults are shown in FIG. 4.

The resulting two amplified DNA fragments were regions of the primersNSJ001P and NSJ002N, i.e., a 3′ terminal portion of the CIN3 DNAfragment and a 5′ terminal portion of the 5′IL DNA fragment, which havepartial homology in the nucleotide sequences. Thus, if PCR is carriedout again using the primers NSX2 and ILSAC, the homologous portions willhybridize so that a fused DNA comprising portions of both genes can beobtained, the homologous portion acting as a linking part (FIG. 5).Accordingly, the recovered CIN3 DNA fragment and 5′IL DNA fragment weremixed and PCR was carried out again using only NSX2 and ILSAC asprimers. The PCR program consisted of 10 cycles of: 92° C. for 1 minute,37° C. for 1 minute and 72° C. for 2 minutes; followed by 20 cycles of:92° C. for 1 minute, 45° C. for 1 minute and 72° C. for 2 minutes;followed by a single cyle of: 92° C. for 1 minute, 55° C. for 1 minuteand 72° C. for 30 minutes. After treatment with phenol and precipitationwith ethanol, 5% polyacrylamide gel electrophoresis was performed toobtain a CNI3IL DNA fragment in which CNI3 and 5′IL were fused. Themethod is shown in FIG. 5.

The CNI3IL DNA fragment was cleaved with the restriction enzyme XhoI andthe resulting fragment was inserted using T4 DNA ligase into pKNI5′which had previously been cleaved with the restriction enzyme XhoI anddephosphorylated with CIAP. The resulting plasmid was cloned into E.coli strain JM 109. In order to select a clone in which the CNI3ILfragment had been inserted in the correct orientation, plasmids wereextracted from the resulting clones and PCR was carried out by using theprimers NSX2 and ILSAC. Using this system, only a DNA band from a clonein which the fragment has been inserted in the correct orientation isdetectable. Plasmid pKSUN9 in which NIa and Mat-IL-11 are ligated in thesame reading frame via a cleavage sequence, Gln-Ala, was obtained (FIG.6).

I) Expression of Ala-IL-11 in a Bacterial Cell

E. coli carrying plasmid pKSUN9 (hereinafter, abbreviated as strainKSUN9) was cultured with shaking at 37° C. overnight in 5 ml of LBmedium containing 42 μg/ml of ampicillin. 2.5 ml of the resulting KSUN9culture was added to 250 ml of fresh LB medium (containing the sameamount of ampicillin) and cultured with shaking at 37° C. until theOD_(600 nm) reached 1.0. Once the OD_(600 nm) had reached 1.0, IPTG wasadded to a final concentration of 0.1 mM and the mixture was culturedwith shaking at 28° C. for a further 12 hours.

A second culture was produced following exactly the same procedure,except that the final culture at 28° C. was performed for 36 hours orlonger with an IPTG concentration of 1 mM, in order to obtain matureIL-11 (which has an N-terminal Pro).

J) Western Blotting

Western blotting was performed in order to determine whether pKSUN9 isfunctional in E. coli and also whether the constructed recombinant geneis expressed.

Each of the 12 and 36 hour cultures induced with IPTG was furtherprocessed as follows. Cells were recovered from 2 ml of the culture bycentrifugation at 3,000×g at 4° C. for 5 minutes, and the pellet wassuspended in 100 μl of 20 mM sodium borate buffer (adjusted to pH 9.0with 0.1 N NaOH). The resulting suspension was treated with a sonicator(Handysonic UR-20P, manufactured by Tomy Seiko Co.) at a setting of 8for 2 minutes to disrupt the cells and then centrifuged at 10,000×g at4° C. for 10 minutes. The supernatant containing the soluble proteinfraction was recovered. Five μl of the supernatant was then run on a 12%SDS polyacrylamide electrophoresis gel following the Laemmli method[Laemmli, U.K. (1970), Nature 227: 680-685].

After electrophoresis, the gel was shaken in a transfer buffer (25 mMTris, 192 mM glycine, 20% methanol) for 5 minutes and blotted onto apiece of PVDF membrane (Trans-Blot Transfer Medium, manufactured byBio-Rad Laboratories) by treatment at 15 V for 1 hour usingTrans-Blot-SD Semi-Dry Transfer Cell (manufactured by Bio-RadLaboratories). The blotted PVDF membrane was washed in PBS-Tw medium for10 minutes. The washed PVDF membrane was transferred to PBS-Twcontaining 5% Skim Milk (manufactured by Snow Brand Milk Products) at37° C. for 1 hour for blocking.

After blocking, the PVDF membrane was further washed in PBS-Tw once for10 minutes and twice for 5 minutes, and then transferred to anti-IL-11rabbit serum which had been diluted 10,000 fold in PBS-Tw, and incubatedat 37° C. for 20 minutes. The PVDF membrane was then washed again withPBS-Tw, once for 10 minutes and twice for 5 minutes.

The PVDF membrane was then transferred to anti-rabbit IgG goat antibodylabeled with horse radish peroxidase (manufactured by Amersham) whichhad been diluted 5,000 fold with PBS-Tw, and incubated at 37° C. for 20minutes. After this time, the PVDF membrane was again washed in PBS-Tw,once for 10 minutes and 4 times, each for 5 minutes.

After this further washing, the PVDF membrane was treated with enhancedchemiluminescence (ECL) detection reagent (manufactured by Amersham),and the bands which reacted with the anti-IL-11 antibody were shown upby bringing the PVDF membrane into contact with an X-ray film for aperiod of between 30 seconds and 5 minutes.

As a result of the above Western blotting, signal bands corresponding tomolecular weights of about 50 kDa and about 23 kDa were detected fromboth the 12-hour and the 36-hour cultures. The 23 kDa band exhibitedsubstantially the same mobility as that of the mature IL-11 used as acontrol. Thus, the 23 kDa signal band appeared to be IL-11 which hadbeen cleaved out of the NIa/IL-11 fusion protein by the proteolyticactivity of NIa at the Gln-Ala linking sequence. It was deduced that theheavier band (50 kDa) was the uncleaved fusion protein.

Hereinafter, the 23 kDa protein obtained from the 12-hour culture willbe referred to as 23 kDa-ON and the 23 kDa protein obtained from the36-hour culture will be referred to as 23 kDa-36 hr.

K) Purification of the 23 kDa-ON and 23 kDa-36 hr Proteins

The 23 kDa-ON and 23 kDa-36 hr proteins were purified in order todetermine the amino acid sequences of their N-termini. 250 ml of each ofthe 12-hour and the 36-hour cultures were obtained by following the sameprocedure described in I) above. Each culture was then further processedas follows. The culture was centrifuged at 5,000×g at 4° C. for 15minutes and the pellet was suspended in 10 ml of 20 mM borate buffer (pH9.0) and disrupted with each of a French press and a sonicator. Thesoluble protein fraction was recovered as the supernatant aftercentrifugation at 15,000×g at 4° C. for 30 minutes.

The resulting soluble protein fraction was then subjected to weak ionexchange column chromatography using FPLC manufactured by Pharmaciausing the following conditions:

Column: CM Toyopearl Pack 650 M (2.2 × 20 cm, manufactured by Toso)Elution buffers: A = 10 mM boric acid-sodium hydroxide (pH 9.0), 13 mMpotassium chloride B = 10 mM boric acid-sodium hydroxide (pH 9.0), 13 mMpotassium chloride, 400 mM sodium chloride Flow rate: 2.5 ml/minFraction volume: 5 ml/tube

The concentration gradient used was a linear gradient from eluent A toeluent B over a period of 120 minutes.

Each eluted fraction was subsequently subjected to an enzyme-linkedimmunosorbent assay (ELISA), to identity fractions containing IL-11.

The ELISA was performed as follows. Each well of a 96 well-immunoplate(Maxisoap; manufactured by Nunc) was loaded with 100 μl of 50 mM sodiumcarbonate buffer (pH 9.6) containing 1 μg/ml of anti-IL-11 mousemonoclonal antibody and the plate was then incubated at 37° C. for 1hour. After this time, each well was washed four times with PBS-T medium(PBS containing 0.1% Tween 20).

Each of the FPLC fractions obtained above was diluted 100 fold withPBS-T and loaded into the wells in aliquots of 100 μl/well. The platewas incubated at 37° C. for 1 hour, the wells were again washed withPBS-T, and then each well was loaded with 100 μl of anti-IL-11 rabbitIgG diluted with PBS-T to a final concentration of 1 μg/ml.

The plate was further incubated at 37° C. for 1 hour and washed withPBS-T, and then each well was loaded with 100 μl of alkalinephosphatase-labeled goat anti-rabbit IgG antibody (manufactured by GibcoBethesda Research Laboratories) which had been diluted 3,000 fold inPBS-T. The plate was again incubated at 37° C. for 1 hour and thenwashed with PBS-T. Each well was then loaded with 100 μl of alkalinephosphatase substrate solution. The plate was incubated at roomtemperature for a further 30 minutes to 1 hour, after which time thecoloring of each well was measured as absorbance at 405 nm to identifythe fraction(s) of interest.

The fractions from the FPLC procedure identified by the above ELISAprocedure as containing a substance reacting with rabbit anti-IL-11antibody (fraction nos. 19 to 25) were pooled, concentrated 100-foldwith Centprep-10 (manufactured by Amicon), and then run on a 12% SDSpolyacrylamide electrophoresis gel in accordance with the Laemmli method(supra). The gel was then electro-blotted onto a piece of PVDF membranein a manner similar to that used in the above in the Western blottingprocedure, but using the Problot membrane (manufactured by AppliedBiosystems) as the PVDF membrane.

After blotting, the membrane was thoroughly washed with redistilledwater, stained with Coomassie Brilliant Blue R-250, destained with 50%methanol, and then the band containing the protein which reacted withanti-IL-11 antibody in the Western blot was excised.

The amino acid sequence of the N-terminus of the protein was analyzedusing a protein sequencer (manufactured by Applied Biosystems). TheN-terminal sequence of the band from 23 kDa-ON which reacted with theanti-IL-11 antibody was determined to be:

Ala-Pro-Gly-Pro-Pro-Pro-Gly-(sequence ID No. 9)

This sequence corresponds to the amino acid sequence −1 to +6 of theamino acid sequence of the mature IL-11 protein. Based on this finding,it could be deduced that the 23 kDa protein obtained from the 12-hourculture and which reacted with anti-IL-11 antibody was Ala-IL-11.Accordingly, it seemed apparent that this protein was generated by theproteolytic activity of NIa cleaving the NIa/IL-11 fusion protein at theGln-Ala site in the specific cleavage sequence.

The N-terminal sequence of the band from 23 kDa-36 hr which reacted withthe anti-IL-11 antibody was determined to be:

Pro-Gly-Pro-Pro-Pro-Gly-Pro-(sequence ID No. 10)

This sequence corresponds to the amino acid sequence +1 to +7 of matureIL-11. Accordingly, we were able to reach the following conclusions.

Following induction with IPTG, the NIa/IL-11 fusion protein wasexpressed in E. coli.

By 12 hours culturing subsequent to induction, the expressed NIa/IL-11fusion protein was cleaved at the Gln-Ala peptide bond in the specificcleavage sequence by the protease activity of NIa.

After cleavage of the peptide bond with NIa, mature IL-11 but having anextra Ala on its N-terminus was expressed in E. coli.

By culturing for a further 24 hours after the expression of Ala-IL-11had been established, Ala-IL-11 matured to IL-11 in which the Alaresidue which had been present at the N-terminus of Ala-IL-11 wasdeleted.

Based on the above findings, it was concluded that the 23 kDa proteinobtained after culturing for 36 hours was a mature type of IL-11 whoseN-terminus was Pro.

Therefore, it was established that IL-11 could be expressed as a fusionprotein with NIa and that the activity of NIa could cleave a specificlinker sequence containing Gln-Ala to afford Ala-IL-11. Continuedculture was then able to afford mature IL-11 wherein the alanine residuehad been deleted to expose a proline N-terminal by a factor present inE. coli.

In order to ensure that the expressed IL-11 and Ala-IL-11 werebiologically active, adipogenesis inhibitory factor (AGIF) activity wasdetermined as an index. The fact that IL-11 has adipogenesis inhibitoryactivity has previously been demonstrated [Kawashima, I. et al. (1991),FEBS L. 283: 199-202].

L) Measuring Inhibitory Effects on the Morphological Changes from 3T3-L1Cells to Adipocytes

The method for determining the AGIF activity used in the presentinvention is as follows. Mouse embryonic fibroblast cell line 3T3-L1[Green, H. and Kehinde, O. (1974), Cell 1: 113-116] purchased from ATCCis used. The cells are in all cases cultured in a mixed humid atmosphereof 10% CO₂-90% air at 37° C. and subcultured with medium A. Induction ofadipogenic differentiation is carried out following the proceduredescribed by Rubin et al. [Rubin, C. S. et al. (1978), J. Biol. Chem.253: 7570-7578].

3T3-L1 cells are suspended in medium A to a density of 1.0×10⁴ cells/ml,seeded onto a 48 well multicluster dish (manufactured by Coaster, 0.5ml/well), and then cultured. After 3 days of culturing, the cells reachconfluence. The medium is then replaced with fresh medium A and, afterculturing for a further 2 days, the medium is replaced with anadipogenesis induction medium, medium B, together with the simultaneousaddition of 0.5 ml of the test sample. The medium is replaced with freshmedium B and a fresh sample every two days.

Instead of medium B, adipocyte maintaining medium, medium C is used toreplace exhausted medium in the wells, starting at varying times fordifferent wells between days 4 and 7 after the first test sample isadded.

After culturing in medium C for 2 days, the cells are fixed with 5%formaldehyde, and any fat particles which have accumulated in the cellsand cell nuclei are stained with Oil Red 0 and hematoxylin,respectively. A micrograph is taken and the number of nuclei and thenumber of cells which have accumulated stained fat particles arecounted. The adipogenetic ratio (AR) is calculated according to thefollowing equation:${{AR}\quad (\%)} = {100 \times \frac{{number}\quad {of}\quad {cells}\quad {accumulating}\quad {fat}\quad {particles}}{{total}\quad {number}\quad {of}\quad {nuclei}}}$

Fixation of the cells and staining with Oil Red 0 and hematoxylin arecarried out in accordance with the procedures described by Yoshio Mitomoand Shojiro Takayama in “Lectures on Clinical Testing” Vol. 12,“Pathology” (1982), published by Ishiyaku Shuppan.

M) Method for the Determination of Lipoprotein Linase InhibitoryActivity

The determination is carried out in accordance with the method describedby Beutler et al. [Beutler, B. A. et al. (1985), J. Immonol. 135:3972-3977]. Adipogenically differentiated 3T3-L1 cells are prepared asdescribed in L) above, except that no test sample is added whendifferentiation into adipocytes is induced. Instead, the test sample isadded together with fresh medium C and the cells are cultured for 18hours.

After this time, the medium is removed and the cells are washed twicewith PBS(−) (phosphate-buffered saline, manufactured by Nissui Seiyaku)and each well is then loaded with 300 μl of medium D and cultured for afurther 1 hour. 100-μl aliquots of each of the culture supernatants aretaken for use in measuring lipoprotein lipase (LPL) activity, which ismeasured in triplicate for each sample to obtain an average.

LPL activity is measured as described by Nilsson-Ehle and Schotz[Nilsson-Ehle, P. and Schotz, M. C. (1976), J. Lipid Res. 17: 536-541].The aliquots of supernatant obtained above are each mixed with an equalvolume of LPL substrate solution and allowed to react at 37° C. for 120minutes. The reaction is stopped by adding 1.05 ml of 0.1 M potassiumcarbonate buffer (adjusted to pH 10.5 with 0.1 M potassium borate) and3.25 ml of a mixture of methanol:chloroform:heptane [141:125:100 (v/v)]with vigorous stirring. The mixture is then centrifuged at 3,000×g for15 minutes at room temperature. The H content of the water-methanolphase is counted with a liquid scintillation counter.

One unit of LPL activity is defined as being that activity whichgenerates 1 μmol of fatty acid per minute. The 13 mM glyceroltri[9,10(n)-³H]oleate in the substrate solution is prepared by dilutingglycerol tri-[9,10(n)-³H]oleate (37.0 GBq/mol), manufactured by Amershamwith triolein (manufactured by Sigma), followed by purification on asilica gel column chromatography.

The following Examples relate to the glutathione reducing proteinembodiment of the present invention.

EXAMPLE 1

Extraction of Poly(A)^(±) RNA from KM-102 Cells

KM-102 cells were cultured in 36 plastic, 15 cm diameter, culture disheswith Iscove's modified minimum essential medium (Boeringer-Mannheim)containing 10% fetal bovine serum. After growing the cells toconfluence, phorbol myristyl acetate (PMA) and calcium ionophore A23187(Sigma) were added to final concentrations of 10 ng/ml and 0.2 μM,respectively, and culturing was continued at 37° C. Lots of 12 disheswere harvested 3, 6 and 14 hours later, and each dish was separatelydissolved in guanidine thiocyanate solution and the liquid phase wascollected.

Isolation of poly(A)⁺ RNA was basically performed as described in“Molecular Cloning—A Laboratory Manual” [Maniatis, T. et al. (1982) pp.196-198]. The following provides a detailed description of theprocedure.

Each recovered liquid phase was individually treated as follows. Theliquid was repeatedly drawn up and discharged from a 10 ml syringebarrel equipped with a 21 G syringe needle. 3 ml of a solution of 5.7 MCsCl-0.1 M EDTA (pH 7.5) was added in advance to a Polyaromar centrifugetube matching the size of a rotor bucket of an RPS40-T centrifuge(Hitachi Koki). The cell preparation was then overlaid on the solutionin the tube until the tube was full.

After centrifuging at 30,000 rpm for 18 hours at 20° C., the resultingpellet was suspended in 400 μl of distilled water followed by ethanolprecipitation. The resulting pellet was dissolved in 400 μl of distilledwater and added to an equal volume of chloroform/1-butanol mixture (4:1v/v) with stirring and the aqueous layer was collected by centrifugalseparation. Ethanol precipitation was performed once again, and theresulting pellet was dissolved in 600 μl of distilled water to obtainwhole RNA. About 4.5 mg of whole RNA was obtained from each of thepooled PMA/A23187-stimulated samples from 3, 6 and 14 hours.

600 μg of each of the three types of whole RNA obtained in this mannerwere pooled and subjected to oligo(dT) cellulose column chromatographyto purify the poly(A)⁺ RNA.

The whole RNA was dissolved in adsorption buffer, and heated at 65° C.for 5 minutes. The resulting solution was applied to an oligo(dT)cellulose column (Pharmacia, type 7) loaded with adsorption buffer, andeluted with eluting solution to recover 100 μg of poly(A)⁺ RNA.

EXAMPLE 2

Preparation of a cDNA Library

A cDNA library was prepared by the Okayama-Berg method.

5 μg of poly(A) ⁺RNA and 24 units of reverse transcriptase (SeikagakuCorp.) were allowed to react at 42° C. for 1 hour in 20 μl of reversetranscriptase reaction solution.

The reaction was stopped by the addition of 2 μl of 0.25 M EDTA and 1 μlof 10% SDS, and the solution was then deproteinized with 20 μl of aphenol/chloroform mixture (1:1 v/v). Following centrifugation to removethe proteinaceous fraction, 20 μl of 4 M ammonium acetate and 80 μl ofethanol were added to the supernatant which was then cooled at −70° C.for 15 minutes. After this time, the precipitate was collected bycentrifugal separation, washed with 75% ethanol and then dried underreduced pressure.

The dried precipitate was dissolved in 15 μl of terminal transferasereaction solution and warmed at 37° C. for 3 minutes. At the end of thistime, 18 units of terminal deoxynucleotidyl transferase (Pharmacia) wereadded to the reaction solution and allowed to react for 5 minutes. 1 μlof 0.25 M EDTA and 0.5 μl of 10% SDS were added to stop the reaction andthe solution was then deproteinized with phenol-chloroform (as describedabove) and centrifuged to remove the proteinaceous fraction. Thesupernatant was collected and thoroughly mixed with 15 μl of 4 Mammonium acetate and 60-1 of ethanol. This mixture was cooled at −70° C.for 15 minutes and the precipitate was collected by centrifugation.

The resulting pellet was dissolved in 10 μl of restriction enzymebuffer, and 2.5 units of restriction enzyme HindIII were added to theresulting solution which was allowed to stand at 37° C. for 1 hour toeffect digestion.

The reaction solution was then deproteinized with phenol-chloroformfollowed by ethanol precipitation, and the supernatant was cooled at−70° C. for 15 minutes. The resulting precipitate was collected bycentrifugation and dissolved in 10 μl of TE buffer [10 mM Tris-HCl (pH7.5) and 1 mM EDTA]. 1 μl of the resulting solution was made up to 10 μlof a reaction solution containing 10 mM Tris-HCl (pH 7.5), 1 mM EDTA,100 mM NaCl and 10 ng of oligo(dG)-tailed linker DNA[3′-oligo(dG)-tailed pL-1 HindIII linker, Pharmacia], followed byheating at 65° C. for 5 minutes and then warming at 42° C. for 30minutes. The reaction mixture was cooled over ice water, followed by theaddition thereto of 10 μl of 10× ligase buffer, 78 μl of distilled waterand 8 units of T4 DNA ligase. The reaction solution was then keptovernight at 12° C.

The following day, the reaction mixture was combined with 10 μl of 1 MKCl, 1 unit of ribonuclease H, 33 units of DNA polymerase I, 4 units ofT4 DNA ligase, 0.5 μl of dNTP solution (20 mM DATP, 20 mM dCTP, 20 mMdGTP and 20 mM dTTP) and 0.1 μl of 50 μg/ml bovine serum albumin (BSA),and the resulting mixture was warmed first at 12° C. for 1 hour and thenat 25° C. for 1 hour. After this time, the reaction solution was dilutedfive-fold with distilled water and was then immediately used totransform E. coli DH5 α using the Hanahan method [Hanahan, D. (1983) J.Mol. Biol. 166, 557-580] thereby to prepare a cDNA library of KM-102cells.

EXAMPLE 3

Preparation of an Oligonucleotide Probe

Based on the AUUUA sequence in the 3′ non-translated region of the mRNAof cytokines, the 15-base oligonucleotide 5′-TAAATAAATAAATAA-3′(sequence ID number 13), designated ATT-3, was chemically synthesized.Synthesis was performed using the 380B automatic DNA synthesizer(Perkin-Elmer Japan Applied Biosystems) following the directionssupplied in the accompanying manual. The method employed was thephosphoamidite method described by Caruthers et al. [Matteucci, M. D.and Caruthers, M. H. (1981) J. Am. Chem. Soc. 103, 3185-3191]. Aftersynthesis of the 15-mer, the resulting oligonuculeotide was severed fromthe support and the protecting groups were removed. The resultingoligonucleotide solution was lyophilized to form a powder which was thendissolved in distilled water and stored frozen at −20° C. until the timeof use.

EXAMPLE 4

Screening the cDNA Library

6,500 colonies generated from the cDNA library prepared in Example 2above were fixed on a nitrocellulose filter in accordance with themethod described by Grunstein and Hogness [Grunstein, M. and Hogness, D.S. (1975) Proc. Natl. Acad. Sci. USA 72, 3961-3965]. The ATT-3 probeprepared in Example 3 was 5′-labelled with ³²P following standardprocedures (see “Molecular Cloning—A Laboratory Manual”), and thelabelled probe was used for colony hybridization.

Pre-hybridization was performed at 37° C. for 3 hours in the following:6×SSC, 1× Denhardt solution, 0.25% SDS, 0.05% sodium pyrophosphate and100 μg/ml of denatured salmon sperm DNA. Hybridization was thenperformed overnight at 31° C. in the following: 6×SSC, 1× Denhardtsolution, 17 μg/ml of yeast tRNA and 0.05% sodium pyrophosphatecontaining the ³²P-labeled probe ATTT-3.

On the following day, the nitrocellulose filter was washed at roomtemperature for 2 hours with a 6×SSC solution containing 0.05% sodiumpyrophosphate. Subsequent autoradiography revealed 33 positive clones.

The plasmid DNA was extracted from the positive clones by followingstandard procedures. Several clones were then selected at random andtheir partial cDNA nucleotide sequences were determined by the dideoxymethod. These partial sequences were then examined for homology withnucleotide sequences registered in the EMBL or GenBank databases via apersonal computer and it was established that some of the partialsequences of clones detected by ATT-3 had homology with parts of the Alurepeat [Schmid, C. W. and Jelinek, W. R. (1982) Science 216, 1065-1070].

A DNA fragment containing the Alu repeat sequence was prepared fromhuman genome DNA and labeled with ³²P, following standard procedures.This labeled DNA was used as a probe in colony hybridization using the33 clones identified above, and it became clear that 12 of the clonespossessed the Alu repeat. The length of the cDNA insert of each of theremaining 21 clones was determined, and it was established that thelength was variable over a range of 50 to 3,600 bases.

Restriction enzyme mapping was performed on the cDNA inserts of theremaining 21 clones, and partial nucleotide sequences were determined asabove. These partial sequences were then examined as above for homologywith nucleotide sequences registered in the EMBL or GenBank databasesvia a personal computer, and those clones having novel sequences wereselected.

EXAMPLE 5

Northern Hybridization of Clone No. 31

One of the clones, clone no. 31 (designated pcD-31) had a cDNA insert ofabout 560 bp. A PstI-AatI fragment (292 bp) was obtained from the cDNAinsert of pcD-31 and was labeled with ³²P for use as a probe in aNorthern blot procedure using poly(A)⁺ RNA prepared from KM-102 cells,following a procedure similar to that of Example 1. This hybridizationwas used to determine the length of the naturally occurring mRNA whichcorresponds to the insert of pcD-31.

The procedure of the Northern hybridization was as follows. 5.5 μg ofpoly(A)⁺ RNA was prepared from KM-102 cells and incubated at 50° C. for1 hour in a mixture of 1 M glyoxal, 50% dimethyl sulfoxide (DMSO) and0.01 M disodium hydrogen phosphate (pH 7.0). At the end of this time, 4μl of electrophoresis pigment were added to the incubated mixture whichwas then electrophoresed on a 1% agarose gel in 1× TAE.

Following the electrophoresis, the RNA on the agarose gel wastransferred overnight onto a nylon membrane filter (Bio Rad, Zeta-Probe)using 20×SSC using the capillary transfer method (see “MolecularCloning—A Laboratory Manual”). After transferrence, the filter wasgently washed with 2×SSC, air dried, and then additionally dried at 80°C. for 2 hours to fix the mRNA.

The PstI-AatI fragment of pcD-31 was labeled with ³²P using theMultiprime DNA Labeling System (Amersham).

Pre-hybridization was performed on the filter for 3 hours at 37° C. in asolution containing 5×SSCP, 2.5× Denhardt solution, 50% formamide, 10 mMdisodium hydrogen phosphate (pH 7.0), 0.5% SDS and 100 μg/ml ofdenatured salmon sperm DNA.

Hybridization was then performed on the filter overnight at 42° C. in asolution containing the ³²P-labeled probe, 5×SSCP, 1× Denhardt solution,50′ formamide, 10 mM disodium hydrogen phosphate (pH 7.0), 0.1% SDS and100 μg/ml of denatured salmon sperm DNA. The following day, the filterfirst was washed for 1 hour at 37° C. with a solution containing 50%formamide, 5×SSC and 0.1% SDS, then washed for 2 hours at the sametemperature with a solution containing 409 formamide, 5×SSC and 0.1%SDS, and finally washed at room temperature for 15 minutes with asolution of 2×SSC. Subsequent autoradiography showed that the cDNAinsert of clone pcD-31 is not full length, and that the full length ofthe corresponding mRNA is 3.9 kb (determined from a calibration curvebased on molecular weight markers).

EXAMPLE 6

Preparing a Fresh Library for Screening Clone pcD-31 cDNA

A fresh cDNA library was prepared using the cDNA Synthesis System Plusand cDNA Cloning System (λgt10, adapter method, supplied by Amersham).

5 μg of poly(A)⁺ RNA extracted from KM-102 cells (following a proceduresimilar to that of Example 1) and 100 units of reverse transcriptasewere reacted at 42° C. for 40 minutes in 50 μl of reverse transcriptasereaction solution. After this time, 20 μCi of [α-³²P]dCTP, 93.5 μl ofsecond strand buffer, 4 units of ribonuclease H and 115 units of DNApolymerase I (all provided with the kit) were added to the reactionsolution which was then first incubated at 12° C. for 1 hour, thenincubated at 22° C. for 1 hour and finally heated at 70° C. for 10minutes. After this heating treatment, 10 units of T4 DNA polymerase(provided with the kit) were added to the reaction solution and allowedto react at 37° C. for 10 minutes.

The reaction mixture was then subjected to phenol-chloroformdeproteinization. The reaction solution was centrifuged and thesupernatant was collected and mixed well with 250 μl of 4M ammoniumacetate and 1 ml of ethanol. The mixture was cooled overnight at −20° C.and the precipitate was collected by centrifugation. The resultingpellet was dissolved in 30 μl of sterile water. 10 μl of the resultingsolution were removed and added to a mixture of 2 μl of ligase/kinasebuffer, 250 μmole of EcoRI adapter and 5 units of T4 DNA ligase (allprovided with the kit) and the resulting mixture was incubated overnightat 15° C.

The whole of the reaction solution was then applied to the sizefractionation column provided with the kit in order to remove the EcoRIadapter. The reaction solution was collected in 120 μl aliquots and eachaliquot was mixed with 200 μl of 0.25× TE buffer. The 10th to 17thfractions were pooled and concentrated with butanol to a total volume of120 μl.

The whole of concentrated prearation was then mixed with 55 μl ofsterile water, 20 μl of ligase/kinase buffer and 40 units of T4polynucleotide kinase (all provided with the kit), and the resultingmixture was incubated at 37° C. for 30 minutes. After this time, thereaction mixture was deproteinized by the phenol-chloroform method threetimes and then precipitated with ethanol and cooled overnight at −20° C.The resulting precipitate was collected by centrifugation and dissolvedin 10 μl of sterile water to provide the cDNA sample.

1 μg of the EcoRI arm of λgt10, 1 μl of ligase/kinase buffer and 2.5units of T4 DNA ligase (all provided with the kit) were added to either2 μl of the cDNA sample, followed by incubation overnight at 15° C. Asample containing 4 μl of the cDNA sample was prepared in the same way.Each sample was then further treated as follows. The whole of theresulting reaction solution was first added to 10 μl of Extract A(provided with kit) and the resulting mixture was then added to 15 μl ofExtract B (provided with kit), and the resulting mixture was incubatedat 20° C. for 20 minutes to allow the in vitro packaging reaction totake place.

After this time, 470 μl of SM buffer were added to the reaction solutionwhich was then stored at 4° C. E. coli strain NM514 treated with 10 mMMgSO₄ was then infected with the stored solution to create a λgt10library of KM-102 cDNA.

EXAMPLE 7

Screening the cDNA Library

2×10⁵ plaques obtained from the combined cDNA libraries prepared inExample 6 were fixed to nylon filters (Hybond N, Amersham) by thefollowing procedure.

Infected E. coli prepared in Example 6 were cultured on ten 9 cm platescontaining solid LB medium so that between 1 and 2×10⁴ plaques wereformed per plate. The plaques were transferred onto the plate by gentlypressing the nylon filter onto the plate. An 18 G syringe needle wasthen used to puncture the filter and mark the gel at 3 locations forreference. The filter was then allowed to stand at 4° C. for 5 to 10minutes and was then peeled off and dipped in an alkaline solution (0.1N NaOH, 1.5 M NaCl) for 20 seconds. The filter was then transferred to aneutral solution [0.2 M Tris-HCl (pH 7.5), 2×SSCP] for a period ofbetween 20 seconds and 1 minute, and was next air-dried at roomtemperature for 2 hours. Finally, the filter was dried at 80° C. for 2hours.

EXAMPLE 8

Probe Preparation and Hybridization

³²P-Labeled probes were created from from pcD-31 (obtained in Example 4)by labeling the PstI-AatI fragment and the EcoT221-AatI fragment (223bp) from pcD-31 using the Multiprime DNA Labelling System. Plaquehybridization was performed on the filter obtained in Example 7 usingthe above probes.

Pre-hybridization was performed by placing the filter in a bath of 50%formamide, 5×SSCP, 2.5× Denhardt solution, 0.01 M disodium hydrogenphosphate (pH 7.0), 0.5% SDS and 100 μg/ml of denatured salmon sperm DNAand incubating at 37° C. for 2 hours.

Hybridization was then performed by placing the filter in a reactionsolution containing the ³²P labeled probes prepared above and 50%formamide, 5×SSCP, 1× Denhardt solution, 0.01 M disodium hydrogenphosphate (pH 7.0), 0.1% SDS and 100 μg/ml of denatured salmon spermDNA, and incubating overnight at 37° C.

The following day, the filter was first washed at room temperature for 3hours with a solution containing 50% formamide, 5×SSC and 0.1% SDS, andthen washed at room temperature for 5 minutes with 2×SSC.Autoradiography showed 80 positive clones obtained in this primaryscreen.

Using the clones identified as positive each time, the procedures ofExamples 7 and 8 were repeated a further three times (quaternaryscreening), and a total of 17 positive clones were ultimately obtained.The cDNA was isolated from each of the 17 clones and the inserts werecut out with EcoRI. The length of each cDNA insert was investigated byagarose gel electrophoresis and clone no. 31-7 was isolated, which had acDNA insert of 3.9 kbp, corresponding to the complete length of theoriginal mRNA.

EXAMPLE 9

Restriction Mapping of Clone No. 31-7

Clone no. 31-7 was digested with EcoRI to isolate and purify the 3.9 kbfragment containing the cDNA insert. This fragment was then insertedinto pUC18 using T4 DNA ligase. E. coli DH5α was transformed with thisnew plasmid. Transformed cells were selected by their resistance toampicillin and clone pUCKM31-7 having a 3.9 kbp cDNA insert wasidentified by digesting the DNA with EcoRI and subjecting the cleavedDNA to agarose gel electrophoresis.

pUCKM31-7 was cleaved with each of the restriction enzymes HindIII,SacI, XbaI, SmaI, BgIII, EcoT22I and AatI, or pairs thereof. Agarose gelelectrophoresis was performed on the resulting fragments and the lengthof each fragment was measured using the λHindIII/φX174HaeIII marker asan indicator. The resulting restriction map is shown in FIG. 7.

EXAMPLE 10

Sequence Determination of Clone No. 31-7

The entire nucleotide sequence of the cDNA insert of pUCKM31-7 wasdetermined by the dideoxy method using an M13 phage. In addition, aportion of the sequence was analyzed with the 373A DNA Sequencer(Perkin-Elmer Japan Applied Biosystems). The resulting nucleotidesequence is ID number 11 in the accompanying Sequence Listing.

The cDNA insert of pUCKM31-7 is 3815 bases long, and clearly has an openreading frame composed of 549 amino acids, starting with methionine. Apoly(A) tail is apparently absent. A comparison of the base sequence ofthe 3′ terminal of the insert of pcD-31 with the sequence of clonepUCKM31-7 reveals that the insert of pUCKM31-7 is only missing thepoly(A) tail portion (FIG. 8), and nothing else.

The EMBL and GenBank nucleotide databases and the NBRF and SWISS-PROTdatabases were accessed in order to compare the base and amino acidsequences, respectively. The closest match which was discovered was a35.3% homology of the peptide sequence with human glutathione reductase.Accordingly, it was concluded that the ORF of the cDNA insert ofpUCKM31-7 clearly encodes a novel polypeptide. This novel polypeptide isshown as sequence ID number 12 in the accompanying Sequence Listing.

EXAMPLE 11

Expression and Purification of the Novel Polypeptide

Construction of a High Expression Vector and Expression in COS-1 Cells

pUCKM31-7 was digested with HindIII and the 3003 bp fragment containingthe cDNA insert was isolated and purified following standard procedures.The terminals of the resulting fragment were blunted using a DNAblunting kit (Takara Shuzo).

Meanwhile, the high expression vector pcDL-SRα296 [Takabe, Y. et al.(1988) Mol. Cell. Biol. 8, 466-472] was digested with PstI and KpnI andthe terminals were blunted using a DNA blunting kit. The blunted insertwas then ligated into the blunted plasmid in a reaction using T4 DNAligase. E. coli was then transformed with the resulting DNA by thecalcium chloride method, and the resulting Amp^(R) transformants wereselected and the plasmid DNA retained by the organisms was analyzed.

Specifically, a strain in which the direction of cDNA transcription wasidentical to the direction of the SRα promoter was selected by digestionof the plasmid with HindIII and BglII followed by agarose gelelectrophoresis to locate an 800 bp fragment, and the plasmid which wasselected was designated pSRα31-7 (FIG. 9). The SRα promoter comprisesthe SV40 initial promoter and the R-U5 sequence of the long terminalrepeat (LTR) of HTLV-1, and has promoter activity which is 10 to 100times stronger than the SV40 initial promoter alone.

Next, COS-1 cells were transfected with the resulting plasmid pSRα31-7.Transfection of COS-1 cells was performed by electroporation using theGTE-1 gene introduction device (Shimadzu).

COS-1 cells were grown to semi-confluence over the bottoms of seven 150cm³ flasks, each containing 25 ml DMEM (containing 10% fetal bovineserum). The cultures were then collected and each was treated with 3 mlof Trypsin-EDTA solution (10× solution available from Sigma) and allowedto stand at room temperature until the cells had separated. 1 ml ofinactivated fetal bovine serum and 9 ml of fresh trypsin-EDTA solutionwere then added and the cells were collected by centrifugation. Thecollected cells were then washed twice with PBS(−) buffer and suspendedin PBS(−) buffer to a density of 6×10⁷ cells/ml.

Meanwhile, plasmid DNA was prepared by the cesium chloride method andmade up to 200 μg/ml in PBS(−) buffer.

20 μl of each of the above-mentioned PBS(−) preparations of cells andplasmid were mixed and the resulting mixture was placed in a chambercontaining electrodes spaced apart at an interval of 2 mm. Two 600Vpulses, each for a duration of 30 μsec were then applied to the mixtureat an interval of 1 second.

The electrode chamber was cooled at 4° C. for 5 minutes and then thecell-DNA mixture inside was mixed with 10 ml of DMEM containing 100fetal bovine serum. This mixture was transferred to a Petri Plate andcultured overnight at 37° C. in a 50% CO₂ atmosphere. The following day,the culture supernatant was discarded, the cells were washed withserum-free DMEM and then suspended in 10 ml of DMEM and cultured at 37°C. for 3 days. After this time, the cell supernatant was harvested.

Culture supernatant was also harvested from the negative control. Thenegative control used the plasmid pcDL-SRα296 containing no cDNA insert,but was otherwise prepared in similar manner to the test culture.

1 ml of each of the culture supernatants of the negative control and thetest culture were separately processed as follows. The supernatant wasfirst treated with trichloroacetic acid (TCA) to precipitate protein,and the precipitate was collected by centrifugal separation. Theresulting precipitate was washed with ice-cooled acetone and air-driedand then dissolved in SDS-polyacrylamide gel electrophoresis (SDS-PAGE)sample buffer containing 2-mercaptoethanol. SDS-PAGE was then performedon a 12.5% gel under reducing conditions.

Silver staining using the silver stain reagent “Daiichi” (DaiichiChemical Detection) was performed on the bands followingelectrophoresis. Several specific bands (molecular weight: about 60,000)from the culture supernatant of the test sample were stained.

Since the molecular weight of the polypeptide encoded in pSRα31-7 isabout 60,000, and it was also deduced from the amino acid sequence thatpost-translational modification to add saccharide side chains wasunlikely, it was concluded that these several specific bandscorresponded to the polypeptide encoded by the cDNA of pSRα31-7.

EXAMPLE 12

Preparation of a High Expression Plasmid for COS-1 Cells

The next step was to verify that the several specific 60 kDa bandsidentified in Example 11 are the same as the polypeptide encoded by theinsert of pSRα31-7. It was also desired to determine the N-terminalamino acid sequence of this polypeptide. Accordingly, a clone wasprepared wherein an extra six His residues were encoded for theC-terminal of the polypeptide before te step codon. Histidine residueshave a high affinity for Ni⁺² and the objective was to express apolypeptide having a histidine hexomer (6×His), which could be purifiedusing an affinity resin column charged with Ni²⁺.

First, a 66 base oligonucleotide5′-CTAGCGCTCTGGGGCAAGCATCCTCCAGGCTGGCTGCCACCACCACCACCACCACTGATCTAGACT-3′(sequence ID No. 14) and the complementary 66 base strand weresynthesized and purified using an Automated DNA Synthesizer 394(Perkin-Elmer Japan Applied Biosystems). Both oligonucleotidepreparations were mixed and incubated at 70° C. for 3 minutes and thenwere additionally warmed at 37° C. for 30 minutes to allow annealing.Subsequently, the terminals were phosphorylated using T4 polynucleotidekinase.

The resulting double-stranded (ds) fragment was ligated using T4 DNAligase into pUCKM31-7 which had previously been digested with Eco47III.The construct is shown in FIG. 10. E. coli DH5α was transformed withthis DNA by the calcium chloride method and the resulting transformedstrains were selected and screened to obtain pUCKM31-7His. It wasconfirmed that there were no abnormalities in the portion ofpUCKM31-7His where the fragment was inserted by analyzing a portion ofthe relevant base sequence of this pUCKM31-7His.

A high-expression plasmid for COS-1 cells was then prepared bysubcloning the insert of pUCKM31-7His into pcDL-SRα296.

pUCKM31-7His was digested with XbaI and HindIII, the fragments werepurified and the terminals of the fragments were blunted using 1 unit ofKlenow fragment in the presence of 2 mM DATP, 2 mM dCTP, 2 mM dGTP, 2 mMdTTP, 50 mM Tris-HCl (pH 7.2), 10 mM MgSO₄, 0.1 mM dithiothreitol and 50μg/ml of BSA.

Meanwhile, the high expression vector pcDL-SRα296 was digested with PstIand KpnI and blunt-ended with a DNA blunting kit. The blunted fragmentwas then ligated into the blunted plasmid using T4 DNA ligase. Theresulting plasmid was then used to transform E. coli DH5α. Transformantswere then selected and screened. A strain in which the direction of cDNAtranscription was identical to the direction of the SRα promoter wasselected, and the plasmid of this strain was designated pSRα31-7His.COS-1 cells were transfected with the resulting plasmid pSRα31-7His andserum-free supernatant was obtained in a manner similar to thatdescribed in Example 11.

EXAMPLE 13

Purification and N-Terminal Amino Acid Sequence Analysis

600 ml of the supernatant obtained in Example 12 were subject todialysis against 17 volumes of dialysis buffer at 4° C. for 15 hours.The buffer was replaced with a further 17 volumes of dialysis buffer anddialysis was continued at 4° C. for an additional 4 hours.

The dialyzed preparation was then subjected to affinity chromatographyusing FPLC (Fast Protein Polynucleotide Liquid Chromatography—Pharmacia)under the following conditions:

Column: 20 ml of ProBond™ Resin (Invitrogen) filled into XK16/20(φ2.0×20 cm, Pharmacia)

Elution buffer:

A) 20 mM phosphate buffer (pH 7.8) containing 200 mM imidazole, 0.5 MNaCl

B) 20 mM phosphate buffer (pH 7.8) containing 300 mM imidazole, 0.5 MNaCl

Flow rate: 1 ml/min

Fraction solution: 5 ml/tube

Elution conditions: After recovering 4 fractions with elution buffer A),16 fractions were recovered with elution buffer B), and the fractionswere numbered in order from 1 to 20.

300 μl of each of the resulting fraction samples were taken andseparately treated subjected to TCA precipitation treatment and theresulting precipitate was prepared and subjected to SDS-PAGE using a12.5% gel under reducing conditions as before. Bands were detected bythe silver stain method and three bands were detected centering aroundfraction no. 10. The existence of 3 bands indicates that the pSRα31-7Hisinsert encodes a polypeptide having 3 different lengths with differentN-terminal sequences.

The remainder of fractions 7 to 14 was concentrated by TCA precipitationand the precipitate was subjected to SDS-PAGE using a 10% gel underreducing conditions. The protein bands were then transferred from thepolyacrylamide gel onto a polyvinylidine difluoride (PVDF) film(ProBlot™, Applied Biosystems) using a gel membrane transfer device(Marisol, KS-8441) operating at 9 V in the presence of transfer buffer[0.02% SDS, 20% methanol, 25 mM Tris-Borate (pH 9.5)] at 4° C. for 2.5hours.

After this time, the membrane was stained with 0.2% naphthol blue black(Sigma), and the three bands corresponding to those previouslyidentified were excised from the membrane, and the sequence of each bandwas determined to the 6th amino acid from the N-terminal using a gasphase protein sequencer (Shimadzu, PPSQ-10). The N terminal of the bandwith the second largest apparent molecular weight (molecular weightabout 60,000) was as follows:

Val-Val-Phe-Val-Lys-Gln (amino acid nos. 1 to 6 of sequence ID no. 12)

These six amino acids correspond to the first six amino acids of the ORFfrom clone 31-7 and also correspond to the sequence of six amino acidsstarting from the 24th amino acid (val) from the N terminal of theprecursor polypeptide encoded by the cDNA inserts of pSRα31-7His andpSRα31-7. Accordingly, deletion of amino acid numbers 1 to 23 from the Nterminal of this precursor polypeptide should result in the secretion ofa mature form of the protein starting with a Val residue.

EXAMPLE 14

Determination of Reducing Activity

i) Construction of an Expression Vector

The polypeptide purified in the previous Examples was only obtainable inextremely small amounts as it was expressed from COS-1 cells. It wasnot, therefore, possible to use the polypeptide for other purposes, suchas activity assays. Accordingly, it was necessary to find a way toexpress the polypeptide encoded by the cDNA insert of pSRα31-7 in analternative host permitting production of suitable quantities forpurification and assaying. To achieve this, the following procedure wasperformed.

pUCKM31-7 was digested with HindIII, the 3003 bp fragment containing thecDNA insert was isolated and purified and the terminals were bluntedusing a DNA blunting kit. The fragment was then further digested withXbaI.

The expression vector pMAL-c [Guan, C. et al. (1987) Gene 67, 21-30] wasdigested with XbaI and StuI, and then the above XbaI-modified HindIIfragment was ligated into this cleaved plasmid using T4 DNA ligase. Theresulting construct is shown in FIG. 11. The construct was then used totransform E. coli TB-1 and Amp^(R) transformants were selected andscreened. A strain in which the direction of cDNA transcription wasidentical to the direction of the promoter was selected, and the plasmidthus obtained was designated pMAL31-7.

ii) Expression and Purification of Fusion Protein

A seed culture of E. coli harboring pMAL31-7 was prepared by culturingwith shaking overnight at 37° C. in 3 ml of LB medium containing 50μg/ml of ampicillin. The following day, 1 ml of the seed culture wasadded to 100 ml of fresh LB culture medium containing 50 μg/ml ofampicillin and cultured with shaking at 37° C. until the OD_(600 nm)reached 0.5. At this stage, IPTG was added to the culture to a finalconcentration of 0.1 mM, and the culture broth was further cultured withshaking overnight at 37° C.

The following day, bacterial cells were recovered from the overnightculture by centrifuging at 6500 rpm for 20 minutes at 4° C. The pelletwas then suspended in 10 ml of column buffer and the cells in theresulting suspension were disrupted by treating with an ultrasonicdisintegrator. Whole cells and cell fragments were then removed bycentrifuging at 8800 rpm for 30 minutes at 0° C., and the solubleprotein fraction was recovered as the supernatant. 1 ml of this solublefraction was then subjected to chromatography on an amylose resin column(New England Biolabs).

The elution buffer for the chromatography was prepared by adding maltoseto 10 ml of the column buffer to a final concentration of 10 mM.

The negative control sample was also chromatographed. This negativecontrol was prepared using a similar procedure, except that the pMAL-cvector was used without any cDNA insert. The reducing activity of theprotein in the chromatography samples was then assayed.

iii) Determination of Reducing Activity

Determination of reducing activity was performed in a cuvette (SARSTEDT,10×4×45 mm) using dichlorophenol-indophenol (DCIP) and oxidizedglutathione.

a) Determination of Reducing Activity Using DCIP

90 μg, as determined using the Protein Assay Kit (Bio-Rad), of each ofthe chromatography samples obtained in ii) above were separately mixedwith 1 ml of 50 μM DCIP (Sigma). 15 μl of 1 mM NADPH(Boehringer-Mannheim) were then added to each of the samples and theOD_(600 nm) and OD_(340 nm) absorbance values were monitored with time.The resulting decrease in absorbance at both wavelengths is shown inFIGS. 12A and 12B and it can be seen that only the pMAL31-7 samplecontains a factor that reduces DCIP.

b) Determination of Reducing Activity Using Oxidized Glutathione

15 ml of 10 mM oxidized glutathione (Boeringer-Mannheim) were added to90.4 μg of each of the chromatography samples obtained in ii) above andwhich had previously been loaded into separate cuvettes. 15 μl of 1 mMNADPH were added to each cuvette, and the absorbance at OD_(340 nm) wasmonitored with time. The results are shown in FIG. 13, and it can beseen that only the protein from the pMAL31-7 sample is capable ofreducing oxidized glutathione. It was also observed that there is noconsumption of NADPH when no oxidized glutathione is present, so that itwas concluded that the protein from the pMAL31-7 sample can only reduceoxidized glutathione in the presence of NADPH.

EXAMPLE 15

Purification and Analysis of N-Terminal Amino Acid Sequence

From Example 13, it was concluded that COS-1 cells transfected withpSRα31-7His expressed a polypeptide having three types of N-terminal.

In a separate experiment, rabbits were immunized with fusion proteinrecovered from E. coli transformed with pMAL31-7 to obtain a polyclonalantibody preparation against KM31-7 protein. Western blotting wasperformed using this polyclonal antibody, and it was clear that threetypes of bands are also detected in the serum-free culture supernatantobtained from COS-1 cells transfected with pSRα31-7. This result issimilar to that obtained in Example 13.

Accordingly, COS-1 cells were transfected with pSRα31-7 with the aim ofcollecting of a large volume of serum-free culture supernatant to allowpurification and analysis of the N-terminal sequence of the KM31-7protein.

COS-1 cells were transfected with pSRα31-7 and were cultured for 3 daysin 150 mm petridishes each containing 30 ml of DMEM. The culturesupernatant was harvested after this time, and 30 ml of fresh mediumwere added to each dish and culture was continued for a further threedays. Once again, the culture supernatant was harvested. Other aspectsof the transfection and culture were as described in Example 11, but 199dishes were cultured.

The harvested supernatants were pooled and 10 liters of serum-freeculture supernatant were collected after centrifugation and this wasdialyzed overnight against 10 mM Tris-HCl (pH 9.0). Ion exchangechromatography was then performed eight times on the dialyzedpreparation under the following conditions using FPLC (Pharmacia):

Column: 20 ml of DEAE Sepharose Fast Flow (Pharmacia) filled intoXK16/20 (φ2.0×20 cm, Pharmacia)

Elution buffers:

A) 10 mM Tris-HCl (pH 9.0)

B) 10 mM Tris-HCl (pH 9.0)−0.5 M NaCl

Flow rate: 1 ml/min

Fraction solution: 3 ml/tube

Elution conditions: Elution buffer A changing over to Elution buffer Bin a linear concentration gradient over a period of 60 minutes.

The fractions eluted at each NaCl concentration from 0.1 M to 0.4 M werecollected and pooled, and dialyzed overnight against a dialysis buffercontaining 0.1 M Tris-HCl, 5 mM EDTA (pH 7.6) and 1 mM2-mercaptoethanol. The dialyzed preparation was then subjected toaffinity chromatography using 2′,5′-ADP Sepharose 4B (Pharmacia) underthe following conditions:

Column: 20 ml of 2′,5′-ADP Sepharose 4B filled into XK16/20 (φ2.0×20 cm,Pharmacia)

Elution buffers:

A) 0.1 M Tris-HCl, 5 mM EDTA (pH 7.6), 1 mM 2-mercaptoethanol

B) 0.1 M Tris-HCl, 5 mM EDTA (pH 7.6), 1 mM 2-mercaptoethanol, 10 mMNADPH

Flow rate: 0.5 ml/min

Fraction solution: 2 ml/tube

Elution conditions: Elution buffer A changing over to Elution buffer Bin a linear concentration gradient over a period of 120 minutes.

100 μl aliquots of each of the resulting fractions were precipitatedwith TCA and the precipitates were subjected to SDS-PAGE using a 12.5%gel under reducing conditions.

After electrophoresis, the gel was silver-stained to detect the proteinbands. Three bands were obtained starting with fraction #11.

The whole of the remainder of fractions #11 to #14 were thenconcentrated by TCA precipitation and the precipitate was subjected toSDS-PAGE using a 12.5% gel under reducing conditions. The protein wasthen transferred from the gel to a ProBlot PVDF membrane (AppliedBiosystems) following electrophoresis. After transfer of the protein tothe membrane, the mebrane was stained with 0.2% naphthol blue black andthe three proteinaceous bands were excised. N terminal sequence analysiswas then performed using a gaseous phase protein sequencer.

The N terminal of the band apparently having the smallest molecularweight of the three types was determined to be Lys-Leu-Leu-Lys-Met.These five amino acids correspond to the five amino acids starting fromthe 49th amino acid from the N terminal of the polypeptide encoded bythe cDNA insert of pSRα31-7. Accordingly, it was concluded that cleavageof the peptide at the 48th residue resulted in one mature form of theprotein starting with an N-terminal Lys.

EXAMPLE 16

Preparation of a Monoclonal Antibody against the KM31-7 Protein

(a) Preparation of Antigen Protein

A seed culture of E. coli harboring pMAL31-7 was prepared by culturing aloop of cells with shaking overnight at 37° C. in 3 ml of LB mediumcontaining 50 μg/ml of ampicillin. 1 ml of the resulting seed culturewas inoculated into 100 ml of fresh LB medium containing 50 μg/ml ofampicillin and this was cultured with shaking at 37° C. until theOD_(600 nm) reached 0.5. At this stage, IPTG was added to the culturebroth a final concentration of 0.1 mM which was then further culturedwith shaking overnight at 37° C.

Cells were recovered from the resulting overnight culture by centrifugalseparation at 6500 r.p.m. for 20 minutes at 4° C., and the pellet wassuspended in 10 ml of column buffer. The cells in the resultingsuspension were disrupted using an ultrasonic disintegrator and theresulting liquid was centrifuged at 8,000 r.p.m. and at 0° C. for 30minutes. The resulting supernatant contained the soluble proteinfraction.

This soluble protein fraction was subjected to chromatography on a 1 mlamylose resin column. Elution was performed with 10 ml of column buffercontaining 10 mM of maltose. The fusion protein obtained from thechromatography was then stored and subsequently used as the antigen.

(b) Preparation of Immunized Mice Spleen Cells

2 ml of Freund's complete adjuvant was added to 2 ml of the antigen(equivalent to 200 μg) purified in a) above to form an emulsion. Thisemulsion was taken up in a 5 ml syringe barrel equipped with a glassjunction, and the emulsion was used to immunize 8 week-old, male BALB/cmice by subcutaneous injection.

Starting with the second round of immunization, Freund's incompleteadjuvant was used as the adjuvant, but following the same procedure aswith the first immunization. Immunization was performed four timesaltogether, at a rate of one immunization roughly every 2 weeks.

Starting with the second immunization, blood was sampled from the venousplexus of the fundus oculi immediately before immunization, and thetiter of anti-KM31-7 antibody in the serum was determined bysolid-phase, enzyme-linked immunosorbent assay (ELISA).

Solid Phase Anti-KM31-7 ELISA

Between 150 and 200 μl (corresponding to about 200 ng of fusion protein)of the serum-free culture supernatant obtained from COS-1 cellstransfected with pSRα31-7 were placed in each well of a 96 well ELISAplate (Costar) for use as the antigen. The plate was then allowed tostand overnight at 4° C. to coat the bottom surfaces of the plate wells.The following day, the plate was washed 3 times with 0.1% Tween20/phosphate buffered saline (0.1 Tween 20/PBS) and then each well wasloaded with 100 μl of BSA prepared to 10 μg/ml with PBS and allowed tostand at room temperature for 1 hour.

After this time, the plate was washed for a further three times with0.1% Tween 20/PBS. 30-100 μl of primary antibody in the form of aserially diluted sample (for example, mouse serum, hybridoma culturesupernatant or monoclonal antibody) were loaded into each well, and theplate was allowed to stand at room temperature for 1 hour.

After this time, the plate was again washed three times with 0.1% Tween20/PBS and then 100 μl of secondary antibody was added to each well. Thesecondary antibody was prepared as a 3000-fold dilution solution of goatanti-mouse IgG-peroxidase complex (Amersham) or a 3000-fold dilutionsolution of goat anti-mouse IgG alkaline phosphatase complex (BIO-RAD).The plate was then allowed to stand at room temperature for 1 to 2hours.

After this time, the plate was again washed three times with 0.1% Tween20/PBS, and then 100 μl of either peroxidase substrate solution(BIO-RAD, Peroxidase Substrate Kit ABTS) or 10% diethanolaminecontaining 0.001% para-nitrophenyl phosphate solution were added to eachwell. The plate was then allowed to stand at room temperature for 15 to30 minutes, whereafter the antibody titer could be calculated bymeasuring the absorbance at 415 nm or 405 nm using a microplate reader(BIO-RAD).

(c) Preparation of Mouse Myeloma Cells

8-Azaguanine-resistant mouse myeloma cells P3-X63-Ag8.653 (653) (ATCCno. CRL-1580) were cultured in a normal medium (complete GIT) to obtaina minimum of 2×10⁷ cells.

(d) Preparation of Hybridoma

1.4×10⁸ immunized mouse spleen cells obtained after the immunizationregimen described in b) above were thoroughly washed with DMEM (NissuiPharmaceutical). The washed cells were then mixed with 1.5×10⁷ mousemyeloma cells P3-X63-Ag8.653 (653) prepared in c) above, and theresulting mixture was centrifuged at 800 r.p.m. for 6 minutes.

The cell group, consisting of a mixture of the spleen cells andP3-X63-Ag8.653 (653) cells, was collected as the pellet and was brokenup. Polyethylene glycol 4000 (polyethylene glycol #4000) as a 50%solution with DMEM was prepared in advance, and this solution wasdripped onto the broken up cells over a period of 1 minute with stirringat a rate of 2 ml/min. DMEM was then added to the cell preparation in asimilar manner for 1 minute at a rate of 2 ml/min. This procedure wasrepeated one more time for both of the polyethylene glycol and DMEMsolutions. Finally, 16 ml of DMEM was added gradually over a period of 3minutes. The resulting cell preparation was then centrifuged at 800r.p.m. for 6 minutes. The resulting supernatant was discarded and thecells were suspended in 35 ml of complete GIT containing 5 to 10 ng/mlof mouse IL-6.

(e) Screening of Hybridomas

100 μl of the suspension prepared in d) above were loaded into each wellof a 96 well plate (Sumitomo Bakelite) which was then cultured at 37° C.in a 7.5% CO₂ incubator. After 7 days of incubation, 50 μl of HAT mediumwere added to each well. After a further 4 days of incubation, another50 μl of HAT medium were added to each well. The plate was thenincubated for 3 more days. After this time, a portion of the culturesupernatant was sampled from wells in which colony growth of fused cellscould be observed, and the titer of anti-KM31-7 antibody was assayed bythe solid-phase ELISA described in b) above. Sampled medium wasimmediately replaced with HT medium.

(f) Cloning

Cloning of cells from wells testing as positive was repeated three timesby limiting dilution analysis. Those clones observed to have aconsistent antibody titer were selected for use as anti-KM31-7monoclonal antibody-producing hybridoma cell lines. At this stage inparticular, ELISA was performed not only as described in b) above, butalso a control ELISA was performed using the serum-free culturesupernatant obtained from COS-1 cells transfected with pcDL-pSRα296 toprepare the solid phase. Accordingly, those cell lines that reacted tothe former but did not react to the control ELISA were selected forcloning.

(g) Purification of Monoclonal Antibody

Culture supernatant from the anti-KM31-7 monoclonal antibody-producinghybridoma cell line was collected, filter sterilized with a 0.22 μmfilter (Millipore), and then the antibody was purified using MAbTrap GII(Pharmacia).

(h) Assaying the Monoclonal Antibody

1) Antigen Specificity of the Monoclonal Antibody

Monoclonal antibodies were confirmed to be specific for KM31-7 proteinby the immune precipitation test using the serum-free culturesupernatant obtained from COS-1 cells transfected with pSRα31-7.

2) Classification of the Monoclonal Antibody

This test was performed using a mouse monoclonal antibody isotyping kit(Amersham), and the antibody was identified as belonging to the IgG1subclass.

EXAMPLE 17

Isolation and Purification of KM31-7 Protein Using an Antigen-AntibodyReaction

This was performed as described in Example 16 h) 1) above. The same testwas also repeated using the antibody and serum-free supernatant obtainedfrom COS-1 cells transfected with pcDL-pSRα296.

1.4 μg of monoclonal antibody was added to 1.7 ml of each of theserum-free supernatants and allowed to react at room temperature for 1hour while centrifuging at 20 r.p.m. in 2.2 ml microcentrifuge tubes.The control was performed using serum-free supernatant obtained fromCOS-1 cells transfected with pSRα31-7 but without adding monoclonalantibody.

30 μl of Protein G Sepharose 4 Fast Flow (Pharmacia), which hadpreviously been washed with 0.1% Tween 20/PBS, were added to each tubeto adsorb the antibody and centrifuging was continued at a speed of 20r.p.m. for 30 minutes at room temperature.

After this time, each mixture was centrifuged for several seconds at10000 r.p.m. in a microcentrifuge, and then the supernatant wascarefully discarded so as not to lose any of the sediment. The pelletswere then individually washed with 0.1% Tween 20/PBS and thenmicrocentrifuged and washed in a similar manner a further 5 times.

The resulting sediment was suspended in SDS-PAGE sample buffer solutioncontaining 10 μl of 2-mercaptoethanol. Each suspension was heated at 90°C. for 2 minutes, and then SDS-PAGE was performed under reducingconditions using a 12.5% gel. Following electrophoresis, the product wastransferred from polyacrylamide gel to a nitrocellulose film (BIO-RAD).Western blotting was performed using the polyclonal anti-KM31-7 antibodydescribed in Example 1, part (a) and the anti-KM31-7 monoclonal antibodywas determined to specifically precipitate KM31-7 protein fromCOS-1/pSRα31-7 serum-free culture supernatant.

EXAMPLE 18

Preparation of CYVV-NIa/KM31-7 Fusion Protein

In order to express KM31-7 protein using the CYVV-NIa proteasetechnique, it is necessary to link the 3′ terminal of the NIa gene inthe same ORF as the KM31-7 protein DNA. Accordingly, the followingtwo-stage procedure was performed.

i) Introduction of the 3′ Side Chain (SmaI-XbaI. 1006 bp) of KM31-7 cDNAinto pKSUN9

In order to obtain a SmaI-XbaI fragment (1,006 bp) containing the 3′ endof KM31-7 cDNA, 7 μg of pSRα31-7 plasmid DNA were digested with therestriction enzymes SmaI and XbaI, and the resulting fragment wascollected and purified using GENECLEAN II (Funakoshi Japan) using a 0.8%agarose gel.

Meanwhile, 5 μg of pKSUN9 plasmid DNA were similarly digested with SmaIand XbaI, and the cleavage fragment was dephosphorylated with bovinealkaline phosphatase (Alkaline Phosphatase E. coli C75, Takara Shuzo,Japan). The resulting dephosphorylated, linearized DNA was ligated withthe SmaI-XbaI KM-31 fragment using a ligation kit (Takara Shuzo), andthe resulting construct was used to transform E. coli strain JM109.Transformants were selected and screened to obtain a clone pNIa31-7SXcontaining a SmaI-XbaI fragment.

ii) Linking of NIa Protease and KM31-7

In order to link the C terminal sequence of NIa with the N-terminalsequence of the KM31-7 sub-type having a Val N-terminal residue in thesame reading frame, four types of polymerase chain reaction (PCR)primers were constructed, using a Perkin-Elmer Japan Applied BiosystemsModel 392 DNA Synthesizer. The primers are as follows:

5′ GGT CAG CAC AAA TTT CCA 3′ (1) 5′ AAA CAC AAC TTG GAA TGA ACA ATT 3′(2) 5′ TCA TTC CAA GTT GTG TTT GTG AAA 3′ (3) 5′ CAT AGG ATG CTC CAA CAA3′ (4)

The first round of PCR was carried out by using 1 μg of pKSUN9 plasmidDNA as the template. 100 pmol each of primers (1) and (2) and 1/10volume of 10-fold concentration Taq polymerase reaction buffer solutionand finally 5 units of Taq polymerase (Takara Shuzo) were added to thereaction solution, in that order. The PCR reaction was first carried outat 72° C. for 3 minutes, followed by 30 cycles of: 94° C. for 1 minute,55° C. for 2 minutes and 72° C. for 3 minutes, and finishing with atreatment at 72° C. for 10 minutes. Following the PCR reaction, theenhanced DNA product was subjected to 8% polyacrylamide gelelectrophoresis. Strips of gel containing DNA were identified byethidium bromide staining and broken up. 300 μl of elution buffer (0.5ammonium acetate, 1 mM EDTA, pH 8.0) were added to each of the crushedstrips and incubated at 37° C. overnight. Centrifuging yielded thesupernatant containing the purified and amplified DNA.

PCR was once again performed in a similar fashion but using 1 μg ofpUCKM31-7 plasmid DNA as the template and using primers (3) and (4), andthe resulting DNA was purified as above.

The amplified fragment from the first PCR contained the sequenceencoding the residues from the N terminal of KM31-7 protein, startingwith Val-Val-Phe, through to 31 bp upstream from the XhoI site of NIa.The amplified fragment from the second PCR contained the sequence codingfor Asn-Cys-Ser-Phe-Gln from the C terminal of NIa through to 32 bpdownstream from the SmaI site of KM31-7 cDNA.

Accordingly, when PCR is performed using both DNA fragments resultingfrom the two PCR's along with primers (1) and (4), the result is ahybridized strand consisting of 9 bp of the 3′ terminal of NIa and 15 bpof the sequence encoding the desired N-terminal of KM31-7. Thus, it ispossible to generate a fused DNA sequence with this portion as the link.

As a result of this logic, the second round of PCR was performed in justthis manner, and the enhanced fragment was collected from the gel.

iii) Introduction of NIa/KM31-7 DNA into pNIa31-7SX

5 μg of the pNIa31-7SX plasmid DNA obtained in i) was digested with XhoIand SmaI, and the resulting DNA was dephosphorylated by treatment withbovine alkaline phosphatase. The PCR product prepared in ii) was alsodigested with XhoI and SmaI and the resulting fragment was then ligatedwith the digested, dephosphorylated pNIa31-7SX using a ligation kit. Theresulting construct was used to transform E. coli strain JM109.

Amp^(R) transformants were then selected and screened. Screening waseffected by digesting with XhoI followed by electrophoresis. Cloneshaving only an 8.0 kbp band were selected. The plasmids of the selectedclones were then digested with HindIII and again electrophoresed. Theplasmid which was selected had a 330 bp band corresponding to part ofthe NIa cDNA insert. This plasmid was designated pNIa31-7V, andcontained the XhoI and SmaI PCR product.

The base sequence of clone pNIa31-7V was determined, and it wasconfirmed that the sequences encoding NIa and KM31-7 were linked in thesame ORF, with the necessary Gln-Val cleavage sequence located betweenNIa and the KM31-7 ptotein.

iv) Production of KM31-7 Protein

Western blotting confirmed that pNIa31-7V was functioning in E. coli andthat the KM-31-7 protein was being expressed by the constructedrecombinant gene.

A seed culture of E. coli harboring the plasmid pNIa31-7V was culturedovernight with shaking in 3 ml of LB medium containing 50 μg/ml ofampicillin. One ml of the seed culture was added to 100 ml of fresh LBmedium containing 50 μg/ml of ampicillin and cultured at 37° C. withshaking until the OD_(600 nm) reached 1.0. At this stage, IPTG was addedto the culture broth to a final concentration of 1 mM, and the culturewas then incubated at 28° C. for two further nights with shaking.

After this time, 1 ml of the culture was transferred to amicrocentrifuge tube and centrifuged for 5 minutes at 15000 rpm. Thesupernatant was discarded and the pellet was mixed with 300 μl ofsterile water and 300 μl of SDS-PAGE sample buffer solution containing2-mercaptoethanol to break up the settled cell bodies. The resultingsuspension was heated at 95° C. for 2 minutes and then 10 μl of thissuspension were subjected to SDS-PAGE on an 8% gel under reducingconditions.

After electrophoresis, the protein was transferred from the gel onto anitrocellulose membrane. This was achieved by contacting the gel withthe membrane and incubating in the presence of a transcription buffersolution (25 mM Tris-HCl, 1.4% glycine and 20% methanol) at 4° C. for2.5 hours and at 19 V using a gel membrane transcription apparatus(Marisol Japan).

The nitrocellulose membrane was then washed with 20 ml of PBS-T medium,and then blocking was performed for 1 hour in 20 ml of PBS-T containing5% skim milk (Snow Brand Co., Ltd). After this time, the membrane wasrinsed with two lots of 20 ml of PBS-T and then allowed to react for 90minutes in 20 ml of PBS-T containing 1 μl of 100-fold dilutedanti-KM31-7 rabbit MAb serum (primary antibody) in sterile water. Thenitrocellulose film was then rinsed once for 15 minutes and then twicefor 5 minutes each with 20 ml of PBS-T.

The washed membrane was then placed in a bath of 3,000-fold dilutedperoxidase-labelled anti-rabbit IgG goat antibody (BIO-RAD) in PBS-T(used as the secondary antibody above), and allowed to stand for 1 hour.The membrane was then washed with 20 ml of PBS-T and transferred into abath of ECL detection reagent (Amersham), and the bands that reactedwith anti-KM31-7 antibody were detected by autoradiography.

Western blotting was performed and a band having a molecular weight ofroughly 60,000 was detected. This band demonstrated the same mobility asthe protein having the second largest molecular weight of the threeKM31-7 proteins detected from the serum-free culture supernatantobtained by transfecting COS-1 cells with pSRα31-7 used as the control.

MEDIA

x M Phosphate Buffer

An x M solution of Na₂HPO₄ adjusted to the desired pH using an x Msolution of NaH₂PO₄.

Inoculation Buffer

0.1 M Tris-HCl buffer, pH 7.0, 0.05 M EDTA, 1% 2-mercaptoethanol.

Extraction Buffer

0.1 M Tris-HCl buffer, 0.05 M EDTA, 1% 2-mercaptoethanol, pH 7.0.

Degradation Solution

200 mM ammonium carbonate, 2% SDS, 2 mM EDTA, 400 μg/ml bentonite and 20μg/ml protease K (pH 9.0).

1×SSC

0.15 M NaCl, 0.015 M trisodium citrate, pH 7.0.

Liquid LB Medium

10 g of Bacto Tryptone (Difco), 5 g of Bacto yeast extract (Difco) and 5g of sodium chloride, all made up to 1 liter with distilled water.

Tris-Calcium Buffer

10 mM Tris, 50 mM calcium choride, adjusted to pH 7.4 with hydrochloricacid.

Lysis Buffer

0.17 g of sucrose, 250 μl of 1 M Tris-HCl buffer (pH 8.0), 200 μl of 0.5M EDTA (pH 8.0), made up to 20 ml with redistilled water.

Alkaline-SDS Solution

0.2 M sodium hydroxide, 1% SDS.

TBE Solution

100 mM Tris, 100 mnM boric acid, 1 mM EDTA.

Denaturation Solution

1.5 M sodium chloride, 0.5 M sodium hydroxide.

Neutralization Buffer

0.5 M Tris, 3 M sodium chloride (pH 7.4).

50× Denhardt's Solution

1% polyvinyl pyrrolidone, 1% bovine serum albumin, 1% Ficoll 400. Thissolution is then diluted with redistilled water, as appropriate, toachieve the desired concentration

5× Denaturation Buffer

125 μl of 1 M glycine (pH 9.0), 25 μl of 1 M magnesium chloride, 850 μlof redistilled water.

5× Labelling Buffer

25 μl of 1 M Tris-HCl buffer (pH 7.9), 5 μl of 1 M magnesium chloride,2.5 μl of 1 M dithiothreitol, 9.2 μl of redistilled water.

10× M9 Salt Solution

0.145 M disodium hydrogenphosphate, 0.172 M potassiumdihydrogeniphosphate, 0.187 M ammonium chloride, 0.137 M sodiumchloride, pH 7.0.

M9 Minimum Agar Medium

10 ml of 10× M9 salt solution, 100 μl of 1 M magnesium sulfate, 1 ml of20% glucose, 50 μl of 1% thiamine hydrochloride salt, 1 ml of 0.01 Mcalcium chloride and 13 ml of redistilled water, all mixed, sterilizedby filtration, and then poured onto plates immediately after theaddition of 50 ml of 3% bactoagar.

Liquid SOB Medium

10 g of bactotryptone, 2.5 g of bactoyeast extract, 100 μl of 5 M sodiumchloride and 125 μl of 1 M potassium chloride are mixed and made up to500 ml with distilled water. After autoclaving, 5 ml of 1 M magnesiumchloride and 5 ml of 1 M magnesium sulfate are added.

TFB1 Buffer

5 ml of 1 M 2-(N-morpholino)ethanesulfonic acid (MES—adjusted to pH 6.2with 1 N HCl), 6.045 g of rubidium chloride, 0.735 g of calcium chloridebihydrate and 4.94 g of manganese chloride tetrahydrate mixed, adjustedto pH 5.8 with glacial acetic acid, made up to 500 ml with redistilledwater and sterilized by filtration.

TFB2 Buffer

1 ml of 1 M 2-(N-morpholino)propanesulfonic acid (MOPS), 1.102 g ofcalcium chloride bihydrate, 0.12 g of rubidium chloride and 15 ml ofglycerol mixed, adjusted to pH 6.5 with glacial acetic acid, made up to100 ml with redistilled water and sterilized by filtration.

Liquid SOC Medium

5 ml of liquid SOB Medium, 90 μl of 20% glucose.

2× YT Medium

16 g of bactotryptone, 5 g of bactoyeast extract and 5 g of sodiumchloride mixed and made up to 1 liter with redistilled water.

PBS-Tw Medium

80 mM disodium hydrogen-phosphate, 20 mM sodium dihydrogenphosphate, 100mM sodium chloride, 0.1% Tween 20.

PBS-T Medium

4 g of sodium chloride, 0.1 g of potassium dihydrogenphosphate, 1.45 gof disodium hydrogenphosphate dodecahydrate, 0.1 g of potassium chlorideand 0.1 g of sodium azide, all made up to 1 liter with redistilledwater, pH 7.4.

Alkaline Phosphatase Substrate Solution

0.01% p-nitrophenyl phosphate dissolved in 10% aqueous diethanolaminesolution which had been adjusted to pH 9.8 with hydrochloric acid.

Medium A

DMEM (Dulbecco's modified Eagle medium, containing 4.5 g/l of glucose),10% inactivated fetal bovine serum (manufactured by Hyclone) and 10 mMHEPES (pH 7.2).

Medium B

DMEM (containing 4.5 g/l of glucose), 10 mM HEPES (pH 7.2), 3%inactivated fetal bovine serum, 5 μg/ml bovine insulin (manufactured bySigma), 8 μg/ml d-biotin (manufactured by Sigma), 4 μg/ml pantothenicacid (manufactured by Sigma), 1.0 μM dexamethasone (manufactured bySigma) and 0.5 mM isobutylmethylxanthine (manufactured by Aldrich).

Medium C

DMEM (containing 4.5 g/l of glucose) containing 5% inactivated fetalbovine serum, 10 mM HEPES (pH 7.2) and 100 ng/ml bovine insulin.

Medium D

DMEM (containing 4.5 g/l of glucose), 5% inactivated fetal bovine serum,10 mM HEPES (pH 7.2), 100 ng/ml bovine insulin and 10 U/ml sodiumheparin (manufactured by Novo Industry Co.).

LPL Substrate Solution

13 mM glycerol tri[9,10(n)-³H]oleate (51.8 KBq/μmol, manufactured byAmersham), 1.3 mg/ml L-α-phosphatidylcholine distearoyl (manufactured bySigma Co.), 20 mg/ml bovine serum albumin (manufactured by Sigma Co.),135 mM Tris hydrochloride [Tris-HCl (pH 8.1), manufactured by SigmaCo.], 16.5% (v/v) glycerol and 16.5% (v/v) inactivated fetal bovineserum.

Guanidine Thiocyanate Solution

4 M guanidine thiocyanate, 1% Sarkosyl, 20 mM ethylenediaminetetraacetic acid (EDTA), 25 mM sodium citrate (pH 7.0), 100 mM2-mercaptoethanol and 0.1% antifoam A (Sigma).

Adsorption Buffer

0.5 M NaCl, 20 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.1% SDS.

Eluting Solution

10 mM Tris-HCl (pH 7.5), 1 mM EDTA and 0.05% SDS.

Reverse Transcriptase Reaction Solution of Example 2

50 ml Tris-HCl (pH 8.3), 8 mM MgCl₂, 30 mM KCl, 0.3 mM dithiothreitol, 2mM dATP, 2 mM dGTP, 2 mM dTTP, 10 μCi [α-³² P]dCTP and 1.4 μg of vectorprimer-DNA (3′-oligo(dT)-tailed pcDV-1, Pharmacia).

Terminal Transferase Reaction Solution

140 mM potassium cacodylate, 30 mM Tris-HCl (pH 6.8), 1 mM CoCl₂, 0.5 mMdithiothreitol, 0.2 μg of poly A and 100 mM dCTP.

Restriction Enzyme Buffer

50 mM NaCl, 10 mM Tris-HCl (pH 7.5), 10 mM MgCl₂ and 1 mMdithiothreitol.

10-Volume Ligase Buffer

10 mM ATP, 660 mM Tris-HCl (pH 7.5), 66 mM MgCl₂ and 100 mMdithiothreitol.

Electrophoresis Pigment

50% glycerol, 0.01 M disodium hydrogen phosphate (pH 7.0) and 0.4%bromophenol blue.

1× TAE

0.04 M Tris-acetate, 0.001 M EDTA.

1×SSCP

120 mM NaCl, 15 mM sodium citrate, 13 mM potassium dihydrogen phosphateand 1 mM EDTA.

Reverse Transcriptase Reaction Solution of Example 6

1× first strand synthesis buffer, 5% sodium pyrophosphate, 100 units ofribonuclease inhibitor, 1 mM dATP, 1 mM dGTP, 1 mM dTTP, 0.5 mM dCTP and3.75 μg of oligo(dT) primer, all provided with the cDNA Cloning System(Amersham).

SM Buffer

100 mM NaCl, 8 mM MgSO₄.7H₂O, 50 mM Tris-HCl (pH 7.5) and 0.01% gelatin.

Dialysis Buffer

20 mM phosphate buffer (pH 7.8) and 0.5 M NaCl.

Column Buffer

10 mM Tris-HCl (pH 7.4), 200 mM NaCl and 1 mM EDTA.

Taq Polymerase Reaction Buffer Solution

Taq containing 500 mM Tris-HCl (pH 8.3), 500 mM KCl, 15 mM MgCl₂, 100 mMDATP, 100 mM dCTP, 100-mM dGTP, 100 mM dTTP and 2 mg/ml of gelatin.

                   #             SEQUENCE LISTING(1) GENERAL INFORMATION:    (iii) NUMBER OF SEQUENCES: 19(2) INFORMATION FOR SEQ ID NO: 1:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 1320 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii) HYPOTHETICAL: N    (iv) ANTI-SENSE: N     (vi) ORIGINAL SOURCE:          (A) ORGANISM: Clover Ye #llow Vein Virus     (ix) FEATURE:          (A) NAME/KEY: CDS           (B) LOCATION: 1..1320          (D) OTHER INFORMATION:     (ix) FEATURE:          (A) NAME/KEY: mat_ #peptide           (B) LOCATION: 10..1311          (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #1:AAG TTC CAA GGG AAA AGT AAG AGA ACA AGA CA#A AAG TTG AAG TTC AGA       48Lys Phe Gln Gly Lys Ser Lys Arg Thr Arg Gl #n Lys Leu Lys Phe Arg  1               5  #                 10  #                 15GCG GCA AGA GAC ATG AAG GAT CGT TAT GAA GT#G CAT GCC GAT GAG GGG       96Ala Ala Arg Asp Met Lys Asp Arg Tyr Glu Va #l His Ala Asp Glu Gly             20      #             25      #             30ACT TTA GTG GAA AAT TTT GGA ACT CGT TAT TC#A AAG AAA GGC AAG ACA      144Thr Leu Val Glu Asn Phe Gly Thr Arg Tyr Se #r Lys Lys Gly Lys Thr         35          #         40          #         45AAA GGT ACT GTT GTG GGT TTG GGT GCA AAA AC#A AGA CGG TTC ACT AAC      192Lys Gly Thr Val Val Gly Leu Gly Ala Lys Th #r Arg Arg Phe Thr Asn     50              #     55              #     60ATG TAT GGT TTT GAC CCC ACG GAG TAT TCA TT#T GCT AGG TAT CTT GAT      240Met Tyr Gly Phe Asp Pro Thr Glu Tyr Ser Ph #e Ala Arg Tyr Leu Asp 65                  # 70                  # 75                  # 80CCA ATC ACG GGT GCA ACA TTG GAT GAA ACC CC#A ATT CAC AAT GTA AAT      288Pro Ile Thr Gly Ala Thr Leu Asp Glu Thr Pr #o Ile His Asn Val Asn                 85  #                 90  #                 95TTG GTT GCT GAG CAT TTT GGC GAC ATA AGG CT#T GAT ATG GTT GAC AAG      336Leu Val Ala Glu His Phe Gly Asp Ile Arg Le #u Asp Met Val Asp Lys        100           #       105           #           110GAG TTA CTT GAC AAA CAG CAC TTA TAC CTC AA#G AGA CCA ATA GAA TGT      384Glu Leu Leu Asp Lys Gln His Leu Tyr Leu Ly #s Arg Pro Ile Glu Cys        115           #       120           #       125TAC TTT GTA AAG GAT GCT GGT CAG AAG GTG AT#G AGG ATT GAT CTA ACA      432Tyr Phe Val Lys Asp Ala Gly Gln Lys Val Me #t Arg Ile Asp Leu Thr    130               #   135               #   140CCC CAC AAC CCA TTG TTG GCA AGC GAT GTT AG#C ACA ACC ATA ATG GGT      480Pro His Asn Pro Leu Leu Ala Ser Asp Val Se #r Thr Thr Ile Met Gly145                 1 #50                 1 #55                 1 #60TAT CCT GAG AGA GAA GGT GAA CTC CGT CAA AC#T GGA AAG GCA AGG TTA      528Tyr Pro Glu Arg Glu Gly Glu Leu Arg Gln Th #r Gly Lys Ala Arg Leu                165   #               170   #               175GTC GAC CCA TCA GAG TTG CCC GCG CGG AAT GA#G GAT ATT GAT GCA GAG      576Val Asp Pro Ser Glu Leu Pro Ala Arg Asn Gl #u Asp Ile Asp Ala Glu            180       #           185       #           190TTT GAG AGT CTA AAT CGC ATA AGT GGT TTG CG#C GAC TAT AAT CCC ATT      624Phe Glu Ser Leu Asn Arg Ile Ser Gly Leu Ar #g Asp Tyr Asn Pro Ile        195           #       200           #       205TCA CAA AAT GTT TGC TTG CTA ACA AAT GAG TC#A GAA GGC CAT AGA GAG      672Ser Gln Asn Val Cys Leu Leu Thr Asn Glu Se #r Glu Gly His Arg Glu    210               #   215               #   220AAG ATG TTT GGA ATT GGA TAT GGT TCA GTG AT#C ATT ACA AAT CAA CAT      720Lys Met Phe Gly Ile Gly Tyr Gly Ser Val Il #e Ile Thr Asn Gln His225                 2 #30                 2 #35                 2 #40CTG TTC AGA AGG AAT AAT GGG GAG TTA TCA AT#T CAA TCC AAG CAT GGC      768Leu Phe Arg Arg Asn Asn Gly Glu Leu Ser Il #e Gln Ser Lys His Gly                245   #               250   #               255TAC TTC AGA TGC CGC AAC ACC ACA AGC TTG AA#G ATG CTG CCT TTG GAG      816Tyr Phe Arg Cys Arg Asn Thr Thr Ser Leu Ly #s Met Leu Pro Leu Glu            260       #           265       #           270GGA CAT GAC ATT TTG TTG ATT CAG TTA CCA AG#G GAC TTT CCA GTG TTT      864Gly His Asp Ile Leu Leu Ile Gln Leu Pro Ar #g Asp Phe Pro Val Phe        275           #       280           #       285CCA CAA AAG ATT CGC TTT AGG GAG CCA AGA GT#G GAT GAC AAA ATT GTT      912Pro Gln Lys Ile Arg Phe Arg Glu Pro Arg Va #l Asp Asp Lys Ile Val    290               #   295               #   300TTG GTC AGC ACA AAT TTC CAG GAA AAG AGT TC#C TCG AGC ACG GTC TCA      960Leu Val Ser Thr Asn Phe Gln Glu Lys Ser Se #r Ser Ser Thr Val Ser305                 3 #10                 3 #15                 3 #20GAG TCC AGT AAC ATT TCA AGA GTG CAG TCA GC#C AAT TTC TAC AAG CAT     1008Glu Ser Ser Asn Ile Ser Arg Val Gln Ser Al #a Asn Phe Tyr Lys His                325   #               330   #               335TGG ATC TCA ACA GTA GCA GGA CAC TGT GGA AA#C CCT ATG GTT TCG ACT     1056Trp Ile Ser Thr Val Ala Gly His Cys Gly As #n Pro Met Val Ser Thr            340       #           345       #           350AAA GAT GGA TTT ATT GTA GGT ATC CAC AGT CT#T GCT TCA TTG ACA GGC     1104Lys Asp Gly Phe Ile Val Gly Ile His Ser Le #u Ala Ser Leu Thr Gly        355           #       360           #       365GAC GTT AAC ATC TTC ACA AGC TTT CCG CCG CA#G TTT GAG AAC AAA TAT     1152Asp Val Asn Ile Phe Thr Ser Phe Pro Pro Gl #n Phe Glu Asn Lys Tyr    370               #   375               #   380CTA CAG AAG CTC AGT GAA CAC ACA TGG TGT AG#T GGA TGG AAA CTA AAT     1200Leu Gln Lys Leu Ser Glu His Thr Trp Cys Se #r Gly Trp Lys Leu Asn385                 3 #90                 3 #95                 4 #00CTT GGA AAG ATT AGT TGG GGT GGA ATC AAC AT#T GTG GAG GAT GCA CCT     1248Leu Gly Lys Ile Ser Trp Gly Gly Ile Asn Il #e Val Glu Asp Ala Pro                405   #               410   #               415GAA GAG CCC TTT ATA ACA TCC AAG ATG GCA AG#C CTT CTT AGT GAT TTG     1296Glu Glu Pro Phe Ile Thr Ser Lys Met Ala Se #r Leu Leu Ser Asp Leu            420       #           425       #           430AAT TGT TCA TTC CAA GCA AGT GCG      #                  #              1320 Asn Cys Ser Phe Gln Ala Ser Ala        435           #       440 (2) INFORMATION FOR SEQ ID NO: 2:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 440 amino #acids           (B) TYPE: amino acid           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein     (vi) ORIGINAL SOURCE:          (A) ORGANISM: Clover Ye #llow Vein Virus     (ix) FEATURE:          (A) NAME/KEY: mat_ #peptide           (B) LOCATION: 4..437          (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #2:Lys Phe Gln Gly Lys Ser Lys Arg Thr Arg Gl #n Lys Leu Lys Phe Arg  1               5  #                 10  #                 15Ala Ala Arg Asp Met Lys Asp Arg Tyr Glu Va #l His Ala Asp Glu Gly             20      #             25      #             30Thr Leu Val Glu Asn Phe Gly Thr Arg Tyr Se #r Lys Lys Gly Lys Thr         35          #         40          #         45Lys Gly Thr Val Val Gly Leu Gly Ala Lys Th #r Arg Arg Phe Thr Asn     50              #     55              #     60Met Tyr Gly Phe Asp Pro Thr Glu Tyr Ser Ph #e Ala Arg Tyr Leu Asp 65                  # 70                  # 75                  # 80Pro Ile Thr Gly Ala Thr Leu Asp Glu Thr Pr #o Ile His Asn Val Asn                 85  #                 90  #                 95Leu Val Ala Glu His Phe Gly Asp Ile Arg Le #u Asp Met Val Asp Lys            100       #           105       #           110Glu Leu Leu Asp Lys Gln His Leu Tyr Leu Ly #s Arg Pro Ile Glu Cys        115           #       120           #       125Tyr Phe Val Lys Asp Ala Gly Gln Lys Val Me #t Arg Ile Asp Leu Thr    130               #   135               #   140Pro His Asn Pro Leu Leu Ala Ser Asp Val Se #r Thr Thr Ile Met Gly145                 1 #50                 1 #55                 1 #60Tyr Pro Glu Arg Glu Gly Glu Leu Arg Gln Th #r Gly Lys Ala Arg Leu                165   #               170   #               175Val Asp Pro Ser Glu Leu Pro Ala Arg Asn Gl #u Asp Ile Asp Ala Glu            180       #           185       #           190Phe Glu Ser Leu Asn Arg Ile Ser Gly Leu Ar #g Asp Tyr Asn Pro Ile        195           #       200           #       205Ser Gln Asn Val Cys Leu Leu Thr Asn Glu Se #r Glu Gly His Arg Glu    210               #   215               #   220Lys Met Phe Gly Ile Gly Tyr Gly Ser Val Il #e Ile Thr Asn Gln His225                 2 #30                 2 #35                 2 #40Leu Phe Arg Arg Asn Asn Gly Glu Leu Ser Il #e Gln Ser Lys His Gly                245   #               250   #               255Tyr Phe Arg Cys Arg Asn Thr Thr Ser Leu Ly #s Met Leu Pro Leu Glu            260       #           265       #           270Gly His Asp Ile Leu Leu Ile Gln Leu Pro Ar #g Asp Phe Pro Val Phe        275           #       280           #       285Pro Gln Lys Ile Arg Phe Arg Glu Pro Arg Va #l Asp Asp Lys Ile Val    290               #   295               #   300Leu Val Ser Thr Asn Phe Gln Glu Lys Ser Se #r Ser Ser Thr Val Ser305                 3 #10                 3 #15                 3 #20Glu Ser Ser Asn Ile Ser Arg Val Gln Ser Al #a Asn Phe Tyr Lys His                325   #               330   #               335Trp Ile Ser Thr Val Ala Gly His Cys Gly As #n Pro Met Val Ser Thr            340       #           345       #           350Lys Asp Gly Phe Ile Val Gly Ile His Ser Le #u Ala Ser Leu Thr Gly        355           #       360           #       365Asp Val Asn Ile Phe Thr Ser Phe Pro Pro Gl #n Phe Glu Asn Lys Tyr    370               #   375               #   380Leu Gln Lys Leu Ser Glu His Thr Trp Cys Se #r Gly Trp Lys Leu Asn385                 3 #90                 3 #95                 4 #00Leu Gly Lys Ile Ser Trp Gly Gly Ile Asn Il #e Val Glu Asp Ala Pro                405   #               410   #               415Glu Glu Pro Phe Ile Thr Ser Lys Met Ala Se #r Leu Leu Ser Asp Leu            420       #           425       #           430Asn Cys Ser Phe Gln Ala Ser Ala         435           #       440(2) INFORMATION FOR SEQ ID NO: 3:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 25 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #3:GTCCATGGGG AAAAGTAAGA GAACA           #                  #               25 (2) INFORMATION FOR SEQ ID NO: 4:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #4:ACTCTGAGAC CGTGCTCGAG             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 5:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 20 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #5:AGGAAAAGAG TTCCTCGAGC             #                  #                   # 20 (2) INFORMATION FOR SEQ ID NO: 6:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 36 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #6:AATTGTTCAT TCCAAGCACC TGGGCCACCA CCTGGC       #                  #       36 (2) INFORMATION FOR SEQ ID NO: 7:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 36 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #7:GCCAGGTGGT GGCCCAGGTG CTTGGAATGA ACAATT       #                  #       36 (2) INFORMATION FOR SEQ ID NO: 8:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 30 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #8:TTGTCAGCAC ACCTGGGAGC TGTAGAGCTC          #                  #           30 (2) INFORMATION FOR SEQ ID NO: 9:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 7 amino  #acids          (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein   (iii) HYPOTHETICAL: N     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #9:Ala Pro Gly Pro Pro Pro Gly 1               5(2) INFORMATION FOR SEQ ID NO: 10:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 7 amino  #acids           (B) TYPE: amino acid          (C) STRANDEDNESS: single           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: protein    (iii) HYPOTHETICAL: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #10:Pro Gly Pro Pro Pro Gly Pro 1               5(2) INFORMATION FOR SEQ ID NO: 11:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 1650 base  #pairs           (B) TYPE: nucleic acid          (C) STRANDEDNESS: double           (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: cDNA to mRNA    (iii) HYPOTHETICAL: N    (iv) ANTI-SENSE: N     (vi) ORIGINAL SOURCE:          (A) ORGANISM: Homo sapi #ens           (H) CELL LINE: KM-102   (vii) IMMEDIATE SOURCE:           (B) CLONE: KM31-7     (ix) FEATURE:          (A) NAME/KEY: CDS           (B) LOCATION: 1..1647          (D) OTHER INFORMATION:     (ix) FEATURE:          (A) NAME/KEY: mat_ #peptide           (B) LOCATION: 70..1647          (D) OTHER INFORMATION:     (ix) FEATURE:          (A) NAME/KEY: sig_ #peptide           (B) LOCATION: 1..69          (D) OTHER INFORMATION:    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #11:ATG TCA TGT GAG GAC GGT CGG GCC CTG GAA GG#A ACG CTC TCG GAA TTG       48Met Ser Cys Glu Asp Gly Arg Ala Leu Glu Gl #y Thr Leu Ser Glu Leu-23         -20         #         -15         #         -10GCC GCG GAA ACC GAT CTG CCC GTT GTG TTT GT#G AAA CAG AGA AAG ATA       96Ala Ala Glu Thr Asp Leu Pro Val Val Phe Va #l Lys Gln Arg Lys Ile         -5          #          1         #       5GGC GGC CAT GGT CCA ACC TTG AAG GCT TAT CA#G GAG GGC AGA CTT CAA      144Gly Gly His Gly Pro Thr Leu Lys Ala Tyr Gl #n Glu Gly Arg Leu Gln 10                  # 15                  # 20                  # 25AAG CTA CTA AAA ATG AAC GGC CCT GAA GAT CT#T CCC AAG TCC TAT GAC      192Lys Leu Leu Lys Met Asn Gly Pro Glu Asp Le #u Pro Lys Ser Tyr Asp                 30  #                 35  #                 40TAT GAC CTT ATC ATC ATT GGA GGT GGC TCA GG#A GGT CTG GCA GCT GCT      240Tyr Asp Leu Ile Ile Ile Gly Gly Gly Ser Gl #y Gly Leu Ala Ala Ala             45      #             50      #             55AAG GAG GCA GCC CAA TAT GGC AAG AAG GTG AT#G GTC CTG GAC TTT GTC      288Lys Glu Ala Ala Gln Tyr Gly Lys Lys Val Me #t Val Leu Asp Phe Val         60          #         65          #         70ACT CCC ACC CCT CTT GGA ACT AGA TGG GGT CT#T GGA GGA ACA TGT GTG      336Thr Pro Thr Pro Leu Gly Thr Arg Trp Gly Le #u Gly Gly Thr Cys Val     75              #     80              #     85AAT GTG GGT TGC ATA CCT AAA AAA CTG ATG CA#T CAA GCA GCT TTG TTA      384Asn Val Gly Cys Ile Pro Lys Lys Leu Met Hi #s Gln Ala Ala Leu Leu 90                  # 95                  #100                  #105GGA CAA GCC CTG CAA GAC TCT CGA AAT TAT GG#A TGG AAA GTC GAG GAG      432Gly Gln Ala Leu Gln Asp Ser Arg Asn Tyr Gl #y Trp Lys Val Glu Glu                110   #               115   #               120ACA GTT AAG CAT GAT TGG GAC AGA ATG ATA GA#A GCT GTA CAG AAT CAC      480Thr Val Lys His Asp Trp Asp Arg Met Ile Gl #u Ala Val Gln Asn His            125       #           130       #           135ATT GGC TCT TTG AAT TGG GGC TAC CGA GTA GC#T CTG CGG GAG AAA AAA      528Ile Gly Ser Leu Asn Trp Gly Tyr Arg Val Al #a Leu Arg Glu Lys Lys        140           #       145           #       150GTC GTC TAT GAG AAT GCT TAT GGG CAA TTT AT#T GGT CCT CAC AGG ATT      576Val Val Tyr Glu Asn Ala Tyr Gly Gln Phe Il #e Gly Pro His Arg Ile    155               #   160               #   165AAG GCA ACA AAT AAT AAA GGC AAA GAA AAA AT#T TAT TCA GCA GAG AGA      624Lys Ala Thr Asn Asn Lys Gly Lys Glu Lys Il #e Tyr Ser Ala Glu Arg170                 1 #75                 1 #80                 1 #85TTT CTC ATT GCC ACT GGT GAA AGA CCA CGT TA#C TTG GGC ATC CCT GGT      672Phe Leu Ile Ala Thr Gly Glu Arg Pro Arg Ty #r Leu Gly Ile Pro Gly                190   #               195   #               200GAC AAA GAA TAC TGC ATC AGC AGT GAT GAT CT#T TTC TCC TTG CCT TAC      720Asp Lys Glu Tyr Cys Ile Ser Ser Asp Asp Le #u Phe Ser Leu Pro Tyr            205       #           210       #           215TGC CCG GGT AAG ACC CTG GTT GTT GGA GCA TC#C TAT GTC GCT TTG GAG      768Cys Pro Gly Lys Thr Leu Val Val Gly Ala Se #r Tyr Val Ala Leu Glu        220           #       225           #       230TGC GCT GGA TTT CTT GCT GGT ATT GGT TTA GA#C GTC ACT GTT ATG GTT      816Cys Ala Gly Phe Leu Ala Gly Ile Gly Leu As #p Val Thr Val Met Val    235               #   240               #   245AGG TCC ATT CTT CTT AGA GGA TTT GAC CAG GA#C ATG GCC AAC AAA ATT      864Arg Ser Ile Leu Leu Arg Gly Phe Asp Gln As #p Met Ala Asn Lys Ile250                 2 #55                 2 #60                 2 #65GGT GAA CAC ATG GAA GAA CAT GGC ATC AAG TT#T ATA AGA CAG TTC GTA      912Gly Glu His Met Glu Glu His Gly Ile Lys Ph #e Ile Arg Gln Phe Val                270   #               275   #               280CCA ATT AAA GTT GAA CAA ATT GAA GCA GGG AC#A CCA GGC CGA CTC AGA      960Pro Ile Lys Val Glu Gln Ile Glu Ala Gly Th #r Pro Gly Arg Leu Arg            285       #           290       #           295GTA GTA GCT CAG TCC ACC AAT AGT GAG GAA AT#C ATT GAA GGA GAA TAT     1008Val Val Ala Gln Ser Thr Asn Ser Glu Glu Il #e Ile Glu Gly Glu Tyr        300           #       305           #       310AAT ACG GTG ATG CTG GCA ATA GGA AGA GAT GC#T TGC ACA AGA AAA ATT     1056Asn Thr Val Met Leu Ala Ile Gly Arg Asp Al #a Cys Thr Arg Lys Ile    315               #   320               #   325GGC TTA GAA ACC GTA GGG GTG AAG ATA AAT GA#A AAG ACT GGA AAA ATA     1104Gly Leu Glu Thr Val Gly Val Lys Ile Asn Gl #u Lys Thr Gly Lys Ile330                 3 #35                 3 #40                 3 #45CCT GTC ACA GAT GAA GAA CAG ACC AAT GTG CC#T TAC ATC TAT GCC ATT     1152Pro Val Thr Asp Glu Glu Gln Thr Asn Val Pr #o Tyr Ile Tyr Ala Ile                350   #               355   #               360GGC GAT ATA TTG GAG GAT AAG GTG GAG CTC AC#C CCA GTT GCA ATC CAG     1200Gly Asp Ile Leu Glu Asp Lys Val Glu Leu Th #r Pro Val Ala Ile Gln            365       #           370       #           375GCA GGA AGA TTG CTG GCT CAG AGG CTC TAT GC#A GGT TCC ACT GTC AAG     1248Ala Gly Arg Leu Leu Ala Gln Arg Leu Tyr Al #a Gly Ser Thr Val Lys        380           #       385           #       390TGT GAC TAT GAA AAT GTT CCA ACC ACT GTA TT#T ACT CCT TTG GAA TAT     1296Cys Asp Tyr Glu Asn Val Pro Thr Thr Val Ph #e Thr Pro Leu Glu Tyr    395               #   400               #   405GGT GCT TGT GGC CTT TCT GAG GAG AAA GCT GT#G GAG AAG TTT GGG GAA     1344Gly Ala Cys Gly Leu Ser Glu Glu Lys Ala Va #l Glu Lys Phe Gly Glu410                 4 #15                 4 #20                 4 #25GAA AAT ATT GAG GTT TAC CAT AGT TAC TTT TG#G CCA TTG GAA TGG ACG     1392Glu Asn Ile Glu Val Tyr His Ser Tyr Phe Tr #p Pro Leu Glu Trp Thr                430   #               435   #               440ATT CCG TCA AGA GAT AAC AAC AAA TGT TAT GC#A AAA ATA ATC TGT AAT     1440Ile Pro Ser Arg Asp Asn Asn Lys Cys Tyr Al #a Lys Ile Ile Cys Asn            445       #           450       #           455ACT AAA GAC AAT GAA CGT GTT GTG GGC TTT CA#C GTA CTG GGT CCA AAT     1488Thr Lys Asp Asn Glu Arg Val Val Gly Phe Hi #s Val Leu Gly Pro Asn        460           #       465           #       470GCT GGA GAA GTT ACA CAA GGC TTT GCA GCT GC#G CTC AAA TGT GGA CTG     1536Ala Gly Glu Val Thr Gln Gly Phe Ala Ala Al #a Leu Lys Cys Gly Leu    475               #   480               #   485ACC AAA AAG CAG CTG GAC AGC ACA ATT GGA AT#C CAC CCT GTC TGT GCA     1584Thr Lys Lys Gln Leu Asp Ser Thr Ile Gly Il #e His Pro Val Cys Ala490                 4 #95                 5 #00                 5 #05GAG GTA TTC ACA ACA TTG TCT GTG ACC AAG CG#C TCT GGG GCA AGC ATC     1632Glu Val Phe Thr Thr Leu Ser Val Thr Lys Ar #g Ser Gly Ala Ser Ile                510   #               515   #               520CTC CAG GCT GGC TGC TGA          #                   #                  #1650 Leu Gln Ala Gly Cys             525(2) INFORMATION FOR SEQ ID NO: 12:      (i) SEQUENCE CHARACTERISTICS:          (A) LENGTH: 549 amino  #acids           (B) TYPE: amino acid          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #12:Met Ser Cys Glu Asp Gly Arg Ala Leu Glu Gl #y Thr Leu Ser Glu Leu-23         -20         #         -15         #         -10Ala Ala Glu Thr Asp Leu Pro Val Val Phe Va #l Lys Gln Arg Lys Ile         -5          #          1         #       5Gly Gly His Gly Pro Thr Leu Lys Ala Tyr Gl #n Glu Gly Arg Leu Gln 10                  # 15                  # 20                  # 25Lys Leu Leu Lys Met Asn Gly Pro Glu Asp Le #u Pro Lys Ser Tyr Asp                 30  #                 35  #                 40Tyr Asp Leu Ile Ile Ile Gly Gly Gly Ser Gl #y Gly Leu Ala Ala Ala             45      #             50      #             55Lys Glu Ala Ala Gln Tyr Gly Lys Lys Val Me #t Val Leu Asp Phe Val         60          #         65          #         70Thr Pro Thr Pro Leu Gly Thr Arg Trp Gly Le #u Gly Gly Thr Cys Val     75              #     80              #     85Asn Val Gly Cys Ile Pro Lys Lys Leu Met Hi #s Gln Ala Ala Leu Leu 90                  # 95                  #100                  #105Gly Gln Ala Leu Gln Asp Ser Arg Asn Tyr Gl #y Trp Lys Val Glu Glu                110   #               115   #               120Thr Val Lys His Asp Trp Asp Arg Met Ile Gl #u Ala Val Gln Asn His            125       #           130       #           135Ile Gly Ser Leu Asn Trp Gly Tyr Arg Val Al #a Leu Arg Glu Lys Lys        140           #       145           #       150Val Val Tyr Glu Asn Ala Tyr Gly Gln Phe Il #e Gly Pro His Arg Ile    155               #   160               #   165Lys Ala Thr Asn Asn Lys Gly Lys Glu Lys Il #e Tyr Ser Ala Glu Arg170                 1 #75                 1 #80                 1 #85Phe Leu Ile Ala Thr Gly Glu Arg Pro Arg Ty #r Leu Gly Ile Pro Gly                190   #               195   #               200Asp Lys Glu Tyr Cys Ile Ser Ser Asp Asp Le #u Phe Ser Leu Pro Tyr            205       #           210       #           215Cys Pro Gly Lys Thr Leu Val Val Gly Ala Se #r Tyr Val Ala Leu Glu        220           #       225           #       230Cys Ala Gly Phe Leu Ala Gly Ile Gly Leu As #p Val Thr Val Met Val    235               #   240               #   245Arg Ser Ile Leu Leu Arg Gly Phe Asp Gln As #p Met Ala Asn Lys Ile250                 2 #55                 2 #60                 2 #65Gly Glu His Met Glu Glu His Gly Ile Lys Ph #e Ile Arg Gln Phe Val                270   #               275   #               280Pro Ile Lys Val Glu Gln Ile Glu Ala Gly Th #r Pro Gly Arg Leu Arg            285       #           290       #           295Val Val Ala Gln Ser Thr Asn Ser Glu Glu Il #e Ile Glu Gly Glu Tyr        300           #       305           #       310Asn Thr Val Met Leu Ala Ile Gly Arg Asp Al #a Cys Thr Arg Lys Ile    315               #   320               #   325Gly Leu Glu Thr Val Gly Val Lys Ile Asn Gl #u Lys Thr Gly Lys Ile330                 3 #35                 3 #40                 3 #45Pro Val Thr Asp Glu Glu Gln Thr Asn Val Pr #o Tyr Ile Tyr Ala Ile                350   #               355   #               360Gly Asp Ile Leu Glu Asp Lys Val Glu Leu Th #r Pro Val Ala Ile Gln            365       #           370       #           375Ala Gly Arg Leu Leu Ala Gln Arg Leu Tyr Al #a Gly Ser Thr Val Lys        380           #       385           #       390Cys Asp Tyr Glu Asn Val Pro Thr Thr Val Ph #e Thr Pro Leu Glu Tyr    395               #   400               #   405Gly Ala Cys Gly Leu Ser Glu Glu Lys Ala Va #l Glu Lys Phe Gly Glu410                 4 #15                 4 #20                 4 #25Glu Asn Ile Glu Val Tyr His Ser Tyr Phe Tr #p Pro Leu Glu Trp Thr                430   #               435   #               440Ile Pro Ser Arg Asp Asn Asn Lys Cys Tyr Al #a Lys Ile Ile Cys Asn            445       #           450       #           455Thr Lys Asp Asn Glu Arg Val Val Gly Phe Hi #s Val Leu Gly Pro Asn        460           #       465           #       470Ala Gly Glu Val Thr Gln Gly Phe Ala Ala Al #a Leu Lys Cys Gly Leu    475               #   480               #   485Thr Lys Lys Gln Leu Asp Ser Thr Ile Gly Il #e His Pro Val Cys Ala490                 4 #95                 5 #00                 5 #05Glu Val Phe Thr Thr Leu Ser Val Thr Lys Ar #g Ser Gly Ala Ser Ile                510   #               515   #               520Leu Gln Ala Gly Cys             525 (2) INFORMATION FOR SEQ ID NO: 13:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 15 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #13:TAAATAAATA AATAA               #                   #                  #    15 (2) INFORMATION FOR SEQ ID NO: 14:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 66 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: double          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #14:CTAGCGCTCT GGGGCAAGCA TCCTCCAGGC TGGCTGCCAC CACCACCACC AC#CACTGATC     60 TAGACT                  #                  #                   #           66 (2) INFORMATION FOR SEQ ID NO: 15:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #15:GGTCAGCACA AATTTCCA              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO: 16:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #16:AAACACAACT TGGAATGAAC AATT           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 17:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 24 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #17:TCATTCCAAG TTGTGTTTGT GAAA           #                  #                24 (2) INFORMATION FOR SEQ ID NO: 18:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 18 base  #pairs          (B) TYPE: nucleic acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear    (ii) MOLECULE TYPE: other nucleic acid,  #synthetic DNA   (iii) HYPOTHETICAL: N     (iv) ANTI-SENSE: N    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #18:CATAGGATGC TCCAACAA              #                   #                  #  18 (2) INFORMATION FOR SEQ ID NO: 19:     (i) SEQUENCE CHARACTERISTICS:           (A) LENGTH: 6 amino  #acids          (B) TYPE: amino acid           (C) STRANDEDNESS: single          (D) TOPOLOGY: linear     (ii) MOLECULE TYPE: protein    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:  #19: Asn Cys Ser Phe Gln Xaa  1               5

What is claimed is:
 1. A polypeptide having the sequence consisting ofamino acid residue numbers 4 to 437 in SEQ ID NO:
 2. 2. A polypeptidewhich consists of amino acid residue numbers 1 to 526 of SEQ ID NO: 12,and which catalyzes the reduction of dichloroindophenol and oxidizedglutathione.
 3. A polypeptide having the sequence consisting of aminoacid residue numbers −23 to 526 of SEQ ID NO: 12.